Method for producing dendritic cells from pluripotent stem cells

• US 9,499,789 B2
• Filed: 02/23/2012
• Issued: 11/22/2016
• Est. Priority Date: 02/23/2011
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Nakahata, Tatsutoshi, Saito, Megumu, Niwa, Akira, Yanagimachi, Masakatsu
• Primary Examiner(s): SCHUBERG, LAURA J
• Application Number:
  US14/001,004
• Publication Number:
  US 20130330822A1
• Time in Patent Office: 1,734 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0639 Dendritic cells, e.g. Langherhans cells in the epidermis

Abstract

A dendritic cell is produced from pluripotent stem cells by culturing pluripotent stem cells by the following steps: (1) performing adherent culture in a medium which comprises a BMP family protein but does not comprise serum; (2) performing adherent culture in a medium which comprises VEGF but does not comprise serum; (3) performing adherent culture in a medium which comprises a hematopoietic factor but does not comprise serum; and (4) performing suspension culture in a medium which does not comprise serum.

Claims

1. A method for producing dendritic cells from mammalian pluripotent stem cells, said method comprising culturing pluripotent stem cells by the steps below:

(1) culturing the pluripotent stem cells in an adherent culture comprising a medium which comprises bone morphogenic protein 4 (BMP4) but does not comprise serum;

(2) culturing the cells obtained by step (1) in an adherent culture comprising a medium which comprises vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and/or stem cell factor (SCF) but does not comprise serum;

(3) culturing the cells obtained by step (2) in an adherent culture comprising a medium which comprises a hematopoietic factor but does not comprise serum, wherein said culturing in step (3) is performed in a first medium comprising hematopoietic factors consisting of (i) SCF, thrombopoietin (TPO), interleukin-3 (IL-3) and Fms-like tyrosine kinase 3 ligand (Flt3-ligand), and then in a second medium comprising hematopoietic factors consisting of (ii) granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF) and Flt3-ligand; and

(4) culturing the cells obtained by step (3) in a suspension culture comprising a medium which comprises GM-CSF and interleukin-4 (IL-4) but does not comprise serum, wherein dendritic cells are produced/collected.

2. The method according to claim 1, wherein said adherent culture in said Steps (1) to (3) is performed in an extracellular matrix-coated dish.

3. The method according to claim 2, wherein said extracellular matrix is a reconstituted basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma cells.

4. The method according to claim 1, wherein the suspension culture medium in Step (4), comprises a medium comprising lipopolysaccharide (LPS) and tumor necrosis factor-α

(TNF-α

).

5. The method according to claim 1, wherein said dendritic cell is positive for HLA-DR and negative for CD14.

6. The method according to claim 5, wherein said dendritic cell is positive for CD83.

7. The method according to claim 1, wherein Step (1) is performed for 4 days.

8. The method according to claim 1, wherein Step (2) is performed for 2 days.

9. The method according to claim 1, wherein said pluripotent stem cell is a human iPS cell.

10. A method for producing dendritic cells from mammalian pluripotent stem cells, said method comprising culturing pluripotent stem cells by the steps below:

(1) culturing the pluripotent stem cells in an adherent culture comprising a medium which comprises bone morphogenic protein 4 (BMP4) but does not comprise serum;

(2) culturing the cells obtained by step (1) in an adherent culture comprising a medium which comprises vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and/or stem cell factor (SCF) but does not comprise serum;

(3) culturing the cells obtained by step (2) in an adherent culture comprising a medium which comprises a hematopoietic factor but does not comprise serum, wherein said culturing in step (3) is performed in a first medium comprising hematopoietic factors consisting of (i) SCF, thrombopoietin (TPO), interleukin-3 (IL-3) and Fms-like tyrosine kinase 3 ligand (Flt3-ligand), and then in a second medium comprising hematopoietic factors consisting of (ii) granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), Flt3-ligand, and SCF; and

(4) culturing the cells obtained by step (3) in a suspension culture comprising a medium which comprises GM-CSF and interleukin-4 (IL-4) but does not comprise serum, wherein dendritic cells are produced/collected.

11. The method according to claim 10, wherein said adherent culture in said Steps (1) to (3) is performed in an extracellular matrix-coated dish.

12. The method according to claim 11, wherein said extracellular matrix is a reconstituted basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma cells.

13. The method according to claim 10, wherein the suspension culture medium in Step (4), comprises a medium comprising lipopolysaccharide (LPS) and tumor necrosis factor-α

(TNF-α

).

14. The method according to claim 10, wherein said dendritic cell is positive for HLA-DR and negative for CD14.

15. The method according to claim 14, wherein said dendritic cell is positive for CD83.

16. The method according to claim 10, wherein Step (1) is performed for 4 days.

17. The method according to claim 10, wherein Step (2) is performed for 2 days.

18. The method according to claim 10, wherein said pluripotent stem cell is a human iPS cell.