Methods for directed differentiation of pluripotent stem cells to immune cells
Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells.
1. A method of producing immune cells comprising:
(a) obtaining induced pluripotent stem cells (iPSCs), wherein the iPSCs are reprogrammed from a population of somatic cells;
(b) incubating the iPSCs in a first defined media comprising a GSK inhibitor, wherein the media is free or essentially free of BMP4, IL-3, Flt3 ligand, and GM-CSF to prepare cells for HPC differentiation;
(c) culturing the cells produced in step (b) in a second defined media comprising BMP4, FGF2, and VEGF to promote mesoderm induction in a plurality of the cells;
(d) culturing the cells produced in step (c) in a third defined media comprising IL-3 and Flt3 ligand, such that a plurality of the cells proliferate and differentiate into HPCs that exhibit increased expression of one or more of CD7, DLL4, CD144, and CD235 as compared to cells produced in step (c); and
(e) culturing the HPCs produced in step (d) under conditions to promote immune cell differentiation, thereby producing immune cells.
2. The method of claim 1 , wherein the immune cells are lymphoid cells.
3. The method of claim 2 , wherein the lymphoid cells are T cells, B cells, and/or NK cells.
4. The method of claim 2 , wherein HPCs that express CD34 and CD43 are cultured under conditions to promote lymphoid differentiation.
5. The method of claim 4 , wherein culturing the HPCs to promote lymphoid differentiation comprises:
(i) culturing HPCs in defined, feeder-free media on a surface coated with matrix and a Notch ligand; and
(ii) maintaining the culture in the presence of one or more cytokines selected from the group consisting of SCF, TPO, IL-7, and Flt-3, thereby producing lymphoid cells.
6. The method of claim 5 , wherein the defined media comprises ascorbic acid and nicotinamide.
7. The method of claim 6 , wherein the ascorbic acid is present at a concentration of 50 μM to 1 mM.
8. The method of claim 6 , wherein the nicotinamide is present at a concentration of 0.1 mM to 5 mM.
9. The method of claim 5 , wherein the matrix is retronectin and the culture further comprises immobilized anti-CD3 antibody.
10. The method of claim 5 , wherein the Notch ligand is DLL4.
11. The method of claim 5 , wherein step (ii) is one to six weeks or two to four weeks.
12. The method of claim 2 , wherein more than 10% of the lymphoid cells are positive for at least two of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4, and CD3.
13. The method of claim 2 , wherein the lymphoid cells retain the T cell receptor gene arrangement of the blood cells.
14. The method of claim 1 , wherein the first defined media comprises blebbistatin and/or Rock inhibitors.
15. The method of claim 1 , wherein the GSK3 inhibitor is CHIR99021.
16. The method of claim 1 , wherein the first defined media further comprises VEGF, and FGF2.
17. The method of claim 1 , wherein the cells are cultured as aggregates for steps (b)-(d) and the cells are individualized prior to step (e).
18. The method of claim 1 , wherein steps (b) to (d) are performed using amine culture plates.
19. The method of claim 1 , wherein the third defined media further comprises one or more of the cytokines selected from the group consisting of IL-3, IL-6, SCF, TPO, and BMP4.
20. The method of claim 1 , wherein the third defined media comprises heparin.
21. The method of claim 1 , wherein the population of somatic cells is a population of blood cells.
22. The method of claims 21 , wherein the population of blood cells comprises T cells.
23. The method of claim 1 , wherein the HPCs differentiate into at least 20 immune cells per input HPC.
24. The method of claim 1 , further comprising isolating CD34 positive cells prior to step (e) by performing magnetic-activated cell sorting (MACS).
25. The method of claim 24 , further comprising isolating cells positive for one or more of CD7, DLL4, CD144, and CD235.