MATURATION OF HEPATOCYTE-LIKE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS

• US 20150307839A1
• Filed: 11/28/2013
• Published: 10/29/2015
• Est. Priority Date: 11/29/2012
• Status: Active Grant

• Current Assignee: Takara Bio Europe AB
• Original Assignee: Takara Bio Europe AB
• Inventors: Edsbagge, Josefina, Kppers-Munther, Barbara
• Granted Patent: US 10,294,457 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/06 Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present invention relates to directed differentiation and maturation of hepatocyte-like cells. In particular, the present invention relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of GSK-3 (Glycogen synthase kinase 3) or activator of Wnt signalling and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The present invention also relates to exposure of hepatocyte-like cells to an activator of a retinoic acid responsive receptor, such as retinoic acid (RA), optionally in combination with an inhibitor of a cycline dependent kinase (CDK) and/or with the overlay of the cells with one or more components characteristic of the mammalian extracellular matrix (matrix overlay). The hepatocyte-like cells obtained in accordance with the present invention show a phenotype which is more similar to that of primary hepatocytes than previously shown.

Claims

1. A method for promoting the maturation of human hepatocyte-like cells, the method comprising:

Exposing said human hepatocyte-like cells to an activator of a retinoic acid responsive receptor.

2. The method according to claim 1, further comprising culturing human hepatic progenitor cells under differentiation conditions to obtain said hepatocyte-like cells.

5. The method according to claim 2, further comprising initially culturing human pluripotent stem (hPS) cells under differentiation conditions to obtain said hepatic progenitor cells.

6. The method according to claim 5, wherein the initial culturing of hPS cells includes culturing the hPS cells under differentiation conditions to obtain cells of the definitive endoderm (DE cells) and further culturing the obtained cells under differentiation conditions to obtain said hepatic progenitor cells.

7. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a GSK3 inhibitor or matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.

8. The method according to claim 1, further comprising exposing said hepatocyte-like cells to an activator of Wnt signaling or matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.

9. The method according to claim 1, further comprising exposing said hepatocyte-like cells to a CDK inhibitor or matrix overlay simultaneous to exposure to the activator of a retinoic acid responsive receptor.

10. The method according to claim 7, further comprising exposing said hepatocyte-like cells to a CDK inhibitor.

11. The method according to claim 5, wherein the differentiating hPS cells are exposed to a DNA demethylating agent.

12. The method according to claim 11, wherein the exposure to said DNA demethylating agent takes place during the differentiation of the hPS cells into DE cells.

13. The method according to claim 2, wherein the hepatic progenitor cells are derived from human pluripotent (hPS) stem cells.

24. The method according to claim 1, wherein the activator of a retinoic acid responsive receptor is a retinoic acid.

34. The method according to claim 7, wherein the GSK-3 inhibitor is selected from the group consisting of:

9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one, also known as Kenpaullone or NSC 664704;

1-Aza-Kenpaullone (9-Bromo-7,12-dihydro-pyrido[3′

,2′

;

2,3]azepino[4,5-b]indol-6(5H)-one);

Alsterpaullone (9-Nitro-7,12-dihydroindolo-[3,2-d][1]benzazepin-6(5)-one);

BIO (2′

Z,3′

£

)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX);

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X);

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII);

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV);

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3beta Inhibitor I);

2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3beta Inhibitor II);

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3beta Inhibitor III);

alpha-4-Dibromoacetophenone (GSK3beta Inhibitor VII);

AR-AO 14418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3beta Inhibitor VIII);

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3beta Inhibitor XI);

TWSI 19 pyrrolopyrimidine compound (GSK3beta Inhibitor XII);

L803 H-KEAPPAPPQSpP-NH2 or its Myristoylated form (GSK3beta Inhibitor XIII);

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);

Aminopyrimidine CHIR99021;

SB216763 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; and

Indirubin-3′

-monoxime.

36. The method according to claim 7, wherein the GSK3 inhibitor further exhibits inhibitory activity towards a cyclin dependent kinase (CDK), such as CDK2.

43. The method according to claim 8, wherein the activator of Wnt signaling is a Wnt protein.

72. The method according to claim 1, wherein said differentiation conditions for obtaining hepatocyte-like cells are characterized by the presence of one or more growth factors or one or more differentiation inducers in the differentiation medium.

3-4. -4. (canceled)

14-23. -23. (canceled)

25-33. -33. (canceled)

35. (canceled)

37-42. -42. (canceled)

44-71. -71. (canceled)

73-78. -78. (canceled)

79. A kit comprising at least one activator of a retinoic acid responsive receptor and at least one item selected from the group consisting of GSK3 inhibitors, activators of Wnt signalling, CDK inhibitors and extracellular matrix (ECM) components or ECM component mixtures.

80. The kit according to claim 79 comprising at least one activator of a retinoic acid responsive receptor, at least one GSK3 inhibitor, and optionally at least one extracellular matrix (ECM) component or ECM component mixture.

81-84. -84. (canceled)

85. A composition comprising at least one activator of a retinoic acid responsive receptor and at least one item selected from the group consisting of GSK3 inhibitors, activators of Wnt signalling and CDK inhibitors.

86. A composition according to claim 85, comprising at least one activator of a retinoic acid responsive receptor and at least one GSK-3 inhibitor, and optionally at least one CDK inhibitor.

87-88. -88. (canceled)