Method for developing natural killer cells from stem cells
A method for producing NK cells from pluripotent stem cells, which includes culturing pluripotent stem cells in a first serum-free medium, aggregating the undifferentiated stem cells to form embryoid bodies, which are cultured to produce hematopoietic precursor cells, and culturing the precursor cells in a serum-free medium to produce the NK cells. Methods for using such NK cells, e.g., in the treatment of cancer and infectious disease are also provided.
1. A method for producing natural killer cells from undifferentiated stem cells, the method comprising:
placing undifferentiated stem cells in a serum-free medium;
aggregating the undifferentiated stem cells in the serum-free medium and forming spin embryoid bodies by spin aggregation;
culturing the spin embryoid bodies in the serum-free medium for inducing production of precursor cells from the spin embryoid bodies;
culturing the precursor cells in a second serum-free medium and in the absence of exogenous stromal cells to produce the natural killer cells from the precursor cells; and
co-culturing the natural killer cells with inactivated artificial antigen presenting cells (aAPCs).
2. A method for producing natural killer cells from pluripotent stem cells, the method comprising:
(a) aggregating the pluripotent stem cells in a first serum-free medium, thereby forming embryoid bodies;
(b) culturing the embryoid bodies in a second serum-free medium, thereby producing hematopoietic progenitor cells; and
(c) culturing the hematopoietic progenitor cells in a third serum-free medium comprising interleukin 3 (IL-3), interleukin 7 (IL-7), interleukin 15 (IL-15), SCF, and Fms-related tyrosine kinase 3 ligand (FLT3L) and in the absence of exogenous stroma or stromal cells, thereby producing natural killer cells; and
(d) co-culturing the natural killer cells with inactivated artificial antigen presenting cell (aAPCs).
3. The method according to claim 2 , wherein the pluripotent stem cells are human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).
4. The method according to claim 2 , wherein the aggregating is performed by spin aggregation.
5. The method according to claim 2 , wherein the second serum-free medium comprises SCF, bone morphogenetic protein 4 (BMP4), and vascular endothelial growth factor (VEGF).
6. The method according to claim 2 , wherein the hematopoietic progenitor cells express CD34.
7. The method according to claim 2 , wherein the hematopoietic progenitor cells co-express CD34 and CD43.
8. The method according to claim 2 , wherein the hematopoietic progenitor cells co-express CD34 and CD45.
9. The method according to claim 2 , wherein the method does not include a step of cell sorting between steps (b) and (c).
10. The method according to claim 2 , wherein the natural killer cells that are produced express one or more of CD56, killer immunoglobulin-like receptors (KIRs), CD16, NKp44, NKp46, and NKG2D.
11. The method according to claim 2 , further comprising co-culturing the natural killer cells with inactivated aAPCs in a medium that comprises interleukin 2 (1L2).
12. An immunotherapeutic method of treating a patient comprising:
(a) producing a population of natural killer (NK) cells by a method of claim 2 ; and
(b) administering an effective amount of said NK cells to a patient in need thereof.
13. The method of claim 12 , wherein the patient is a cancer patient.
14. The method of claim 13 , wherein the patient has an infectious disease.
15. The method of claim 14 , wherein the patient has a viral infection.
16. The method of claim 12 , wherein the pluripotent cells are from the patient.
17. An immunotherapeutic method of treating a patient comprising administering an effective amount of natural killer cells produced by a method of claim 2 to a patient in need thereof.