Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts

• US 8,822,218 B2
• Filed: 12/01/2010
• Issued: 09/02/2014
• Est. Priority Date: 12/04/2009
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Stem Cell & Regenerative Medicine International, Inc. (Astellas Pharma Incorporated)
• Inventors: Kimbrel, Erin, Lu, Shi-Jiang
• Primary Examiner(s): Cochrane Carlson, Karen
• Assistant Examiner(s): MOSS, NATALIE M
• Application Number:
  US13/512,795
• Publication Number:
  US 20120282693A1
• Time in Patent Office: 1,371 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 39/4613 Natural-killer cells [NK or NK-T]
• CPC Class Codes: A61K 39/4644 Cancer antigens
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2304 Interleukin-4 (IL-4)
• 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 5/0639 Dendritic cells, e.g. Langherhans cells in the epidermis
• CPC Class Codes: C12N 5/0646 Natural killers cells [NK], NKT cells

Abstract

This invention provides methods of generating natural killer (NK) cells and dendritic cells (DCs). The methods utilize human hemangioblasts as intermediate cells to generate the NK cells and DCs. In various embodiments, the methods do not require the use of stromal feeder layers.

Claims

1. A method to generate dendritic cells (DCs), comprising:

providing hemangioblasts;

culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF;

adding IL4 to the liquid media; and

further culturing the hemangioblasts to generate the DCs.

2. The method of claim 1, wherein culturing the hemangioblasts is for about 7 to 11 days.

3. The method of claim 1, wherein further culturing of the hemangioblast after the addition of IL4 is for about 8 to 10 days.

4. The method of claim 1, further comprising adding a cytokine mixture comprising IL1b, TNFα

and IL6 to induce maturation of the DCs.

5. The method of claim 4, wherein the cytokine mixture is added for about 48 hours.

6. The method of claim 4, wherein the cytokine mixture further comprises a cytokine selected from the group consisting of PGE2, IFNα

2b, poly I;

C, IFNγ and

combinations thereof.

7. The method of claim 1, further comprising adding LPS, IFNγ

and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs.

8. The method of claim 1, wherein the concentration of SCF is about 20-100 ng/ml, FL is about 10-50 ng/ml, IL3 is about 5-50 ng/ml, GM-CSF is about 50-100 ng/ml, and IL4 is about 50-100 ng/ml.

9. The method of claim 4, wherein the concentration of IL1b is about 10 ng/ml, TNFγ

is about 10 ng/ml, and IL6 is about 150 ng/ml.

10. The method of claim 6, wherein the concentration of PGE2 is about 1 μ

g/ml, IFNα

2b is about 3000 units/ml, poly I;

C is about 20 μ

g/ml, and IFNγ

is about 20 ng/ml.

11. The method of claim 1, wherein the DCs are mature DCs and express CD83.

12. The method of claim 1, wherein the DCs are mature DCs and the expression of CD209, HLA DR and/or CD11c is increased.