Method for inducing differentiation of human pluripotent stem cells into intermediate mesoderm cells

• US 9,890,357 B2
• Filed: 12/19/2012
• Issued: 02/13/2018
• Est. Priority Date: 12/19/2011
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Osafune, Kenji, Araoka, Toshikazu
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US14/366,169
• Publication Number:
  US 20140363888A1
• Time in Patent Office: 1,882 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/415 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0686 Kidney cells

Abstract

The present invention provides a method for inducing differentiation of a human pluripotent stem cell into an intermediate mesoderm cell without the use of a growth factor. Such method comprises performing culture in a medium containing a specific compound, so as to induce differentiation of a human pluripotent stem cell into an intermediate mesoderm cell.

Claims

1. A method for producing an intermediate mesoderm cell from a human pluripotent stem cell, the method comprising:

(i) culturing the human pluripotent stem cell in a medium comprising a GSK-3 beta inhibitor, or a GSK-3 beta inhibitor and at least one of AM580 and TTNPB; and

(ii) culturing the cell cultured in the culturing (i) in a medium comprising at least one of AM580 and TTNPB to produce the intermediate mesoderm cell.

2. The method of claim 1, wherein the GSK-3 beta inhibitor is CHIR99021.

3. The method of claim 2, wherein the medium in the culturing (i) comprises the GSK-3 beta inhibitor and one of AM580 and TTNPB, and the medium in the culturing (ii) comprises one of AM580 and TTNPB.

4. The method of claim 1, wherein the medium in the culturing (i) comprises the GSK-3 beta inhibitor and one of AM580 and TTNPB, and the medium in the culturing (ii) comprises one of AM580 and TTNPB.

5. The method of claim 1, wherein the medium in the culturing (i) comprises the GSK-3 beta inhibitor and at least one of AM580 and TTNPB.

6. The method of claim 1, wherein the human pluripotent stem cell is a human iPS cell or a human ES cell.

7. The method of claim 1, wherein a culture period of the culturing (i) is up to 2 days and a culture period of the culturing (ii) is at least 3 days.

8. The method of claim 7, wherein the culture period of the culturing (i) is 2 days and the culture period of the culturing (ii) is 8 days.

9. The method of claim 1, wherein a composition of the medium in the culturing (i) is different from a composition of the medium in the culturing (ii).

10. The method of claim 1, wherein the culturing (ii) further comprises assaying the cell cultured in the culturing (ii) for OSR1 such that an OSR1-positive cell is selected as the intermediate mesoderm cell.

11. A method for producing luminal structure constructed with renal tubule cells, the method comprising:

producing an intermediate mesoderm cell by the method of claim 1; and

forming renal tubule cells by suspension culturing the intermediate mesoderm cell such that a sphere consisting of the intermediate mesoderm cell is formed.

12. A method for producing luminal structure constructed with renal tubule cells, the method comprising:

producing an intermediate mesoderm cell by the method of claim 1; and

forming renal tubule cells by culturing the intermediate mesoderm cell with a metanephric cell obtained from a mouse embryo.

13. The method of claim 12, wherein the metanephric cell is obtained from an E11.5 mouse embryo.

14. A method for producing a plurality of intermediate mesoderm cells from a plurality of human pluripotent stem cells, the method comprising:

(i) dissociating the plurality of human pluripotent stem cells into single cells and culturing the dissociated human pluripotent stem cells in a medium comprising a GSK-3 beta inhibitor, or a GSK-3 beta inhibitor and at least one of AM580 and TTNPB; and

(ii) culturing the cells cultured in the culturing (i) in a medium comprising at least one of AM580 and TTNPB to produce the plurality of intermediate mesoderm cells.

15. The method of claim 14, wherein the medium in the culturing (i) further comprises a ROCK inhibitor.

16. The method of claim 15, wherein the ROCK inhibitor is Y-27632.

17. The method of claim 15, wherein the medium in the culturing (i) comprises the GSK-3 beta inhibitor, at least one of AM580 and TTNPB, and the ROCK inhibitor.

18. A method for producing a metanephric cell, the method comprising:

inducing differentiation of a human pluripotent stem cell into an intermediate mesoderm cell by (i) culturing the human pluripotent stem cell in a medium comprising a GSK-3 beta inhibitor, or a GSK-3 beta inhibitor and at least one of AM580 and TTNPB, and (ii) culturing the cell cultured in the culturing (i) in a medium comprising at least one of AM580 and TTNPB; and

inducing differentiation of the intermediate mesoderm cell by adhesion culturing the intermediate mesoderm cell to produce the metanephric cell.

19. The method of claim 18, wherein the metanephric cell comprises at least one selected from the group consisting of a metanephric mesenchyme cell, a metanephric stroma cell, an ureteric bud cell, podocyte, and a proximal tubule cell.

20. The method of claim 18,

wherein the adhesion culturing of the intermediate mesoderm cell occurs in a medium comprising at least one of Wnt3a and BMP7.