COMPOSITIONS AND METHODS FOR PROMOTING THE GENERATION OF DEFINITIVE ENDODERM

• US 20120088300A1
• Filed: 02/05/2010
• Published: 04/12/2012
• Est. Priority Date: 02/06/2009
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Melton, Douglas A., Borowiak, Malgorzata, Maehr, Rene, Chen, Shuibing, Tang, Weiping, Schreiber, Stuart L., Fox, Julia L.
• Granted Patent: US 8,507,274 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

Certain embodiments disclosed herein are directed to a method of producing endoderm cells, such as definitive endoderm cells by exposing stem cells such as embryonic stem cells or induced pluripotent stem (iPS) cells to an effective amount of at least one compound described herein to differentiate the stem cells into the endoderm cells such as definitive endoderm cells. Differentated endoderm cells produced by the methods disclosed herein can be differentiated into pancreatic epithelium, and other endoderm derivatives such as thymus, liver, stomach, intestine and lung. Another aspect of the present invention relates to a method of producing pancreatic progenitor cells, such as Pdx1-positive pancreatic progenitor cells by exposing endoderm cells, such as definitive endoderm cells to an effective amount of at least one compound described herein to differentiate the definitive endoderm cells into Pdx1-positive pancreatic progenitor cells. Kits and compositions comprising Pdx1-positive pancreatic progenitor produced using the methods are also described.

Claims

1. A method of producing a definitive endoderm cell from a pluripotent stem cell comprising contacting a population of pluripotent stem cells with at least one compound of Formula (I) to induce the differentiation of at least one pluripotent stem cell into a definitive endoderm cell, wherein the definitive endoderm cell expresses Sox17, or HNF3B (FoxA2), or Sox17 and HNF3B (FoxA2) and wherein the compound of formula (I) is:

2. The method of claim 1, wherein the pluripotent stem cell is an embryonic stem (ES) cell or an induced pluripotent stem (iPS) cell.

4. The method of claim 1, wherein the stem cell is from a mammal.

5. The method of claim 1, wherein the stem cell is a human stem cell.

6. The method of claim 1, wherein the method further comprising isolating a population of definitive endoderm cells, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater of the total cells in the isolated population are definitive endoderm cells.

7. The method of claim 1, wherein the compound of formula (I) is 2-[(6-carboxy-hexanoyl)-hydrazonomethyl]-benzoic acid (IDE1) or 7-(2-cyclopentylidenehydrazino)-7-oxoheptanoic acid (IDE2).

8. The method of claim 1, wherein the compound of formula (I) has the structure

19. The method of claim 1, wherein at least 20% of the pluripotent stem cells in the population of pluripotent stem cells are induced to differentiate a definitive endoderm cell.

20. The method of claim 1, wherein at least 40% of the pluripotent stem cells in the population of pluripotent stem cells are induced to differentiate a definitive endoderm cell.

21. The method of claim 1, wherein at between 80-90% of the pluripotent stem cells in the population of pluripotent stem cells are induced to differentiate a definitive endoderm cell.

22. The method of claim 1, the method further comprising exposing the stem cells to at least one additional agent.

23. The method of claim 22, wherein the additional agent is selected from the group consisting of:

Nodal, Activin A or Wnt3a.

31. The method of claim 1, further comprising differentiating the definitive endoderm cell into a Pdx1-positive pancreatic progenitor cell, wherein the Pdx1-positive pancreatic progenitor cell expresses Pdx1.

32. The method of claim 31, wherein a Pdx1-positive pancreatic progenitor cell also expresses HNF6.

33. The method of claim 31, comprising contacting a population of definitive endoderm cells with at least one compound of Formula (II) to induce the differentiation of at least one definitive endoderm cell into a Pdx1-positive pancreatic progenitor cell, wherein the compound of formula (II) is:

34. The method of claim 33, wherein the compound of Formula (II) is (2S,5S)-1,2,4,5,6,8-Hexahydro-5-(hydroxymethyl)-1-methyl-2-(1-methylethyl)-3H-pyrrolo[4,3,2-gh]-1,4-benzodiazonin-3-one ((−

)-indolactam V).

35. The method of claim 31, further comprising isolating the population of Pdx1-positive pancreatic progenitor cells.

36. The method of claim 31, further comprising differentiating the population of Pdx1-positive pancreatic progenitor cells into a population of insulin producing cells.

37. The method of claim 31, further comprising differentiating the population of Pdx1-positive pancreatic progenitor cells into a population of cells having at least one characteristic of endogenous pancreatic β

-cells.

38. The method of claim 37, wherein a cell with at least one characteristic of an endogenous pancreatic β

-cell is secretion of insulin in response to glucose.

3. (canceled)

9-18. -18. (canceled)

24-30. -30. (canceled)

39-82. -82. (canceled)

83. A compound of formula (I):

84. The compound of claim 83, wherein R¹ is H, methyl, ethyl, or propyl.

85. The compound of claim 83, wherein R² is H, methyl, ethyl, or propyl.

86. The compound of claim 83, wherein L is —

(CH₂)₅—

.

87. The compound of claim 83, wherein the compound of formula (I) is 7-(2-cyclopentylidenehydrazino)-7-oxoheptanoic acid (IDE2).

88. The compound of claim 87, wherein 7-(2-cyclopentylidenehydrazino)-7-oxoheptanoic has the structure:

89. A compound having the name 2-[(6-carboxy-hexanoyl)-hydrazonomethyl]-benzoic acid (IDE1).

90. The compound of claim 89, wherein 2-[(6-carboxy-hexanoyl)-hydrazonomethyl]-benzoic acid has the structure: