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 for providing an enriched population of lymphoid progenitors comprising:
(a) obtaining a starting cell population comprising lymphoid progenitors;
(b) enriching said starting cell population for lymphoid progenitors by performing magnetic-activated cell sorting (MACS) to isolate cells positive for at least two of the cell surface markers selected from the group consisting of CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4, thereby providing a population of cells with increased lymphoid potential as compared to an unsorted cell population or a population of cells negative for two more of the cell surface markers selected from the group consisting of CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4, wherein cells with increased lymphoid potential have an increased efficiency at producing T cells as measured by a ratio of input of HPCs to output of T cells.
2. The method of claim 1 , wherein step (b) comprising isolating cells positive for CD144 and CD34.
3. The method of claim 1 , wherein step (b) comprising isolating cells positive for CD144 and CD45.
4. The method of claim 1 , wherein step (b) comprising isolating cells positive for CD144 and CD7.
5. The method of claim 1 , wherein step (b) comprising isolating cells positive for CD144, CD34, CD45, and CD7.
6. The method of claim 1 , wherein obtaining the starting cell population comprises culturing hematopoietic precursor cells (HPCs) under conditions to promote lymphoid differentiation.
7. The method of claim 6 , wherein the HPCs are obtained by:
(a) culturing induced pluripotent stem cells (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;
(b) culturing the cells produced in step (a) in a second defined media comprising BMP4, FGF2, and VEGF sufficient to promote mesoderm induction in a plurality of the cells; and
(c) culturing the cells in step (b) in a third defined media comprising IL-3 and Flt3 ligand, such that a plurality of the cells proliferate and differentiate into HPCs.
8. The method of claim 7 , further comprising isolating HPCs positive for at least two of the cell surface markers selected from the group consisting of CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4.
9. The method of claim 1 , wherein method is performed in serum free conditions.
10. The method of claim 1 , further comprising culturing the population of cells with increased lymphoid potential to promote lymphoid differentiation.
11. The method of claim 10 , wherein culturing the cells to promote lymphoid differentiation comprises:
(i) culturing HPCs in defined media on a surface coated with matrix and a Notch ligand, wherein the HPCs express one or more of the cell surface markers selected from the group consisting of CD34, CD43, CD7, DLL4, CD144, and CD235; and
(ii) maintaining the culture in the presence of one or more cytokines, thereby producing lymphoid cells.
12. The method of claim 11 , wherein the matrix is extracellular matrix protein.
13. The method of claim 12 , wherein the matrix is retronectin, collagen, laminin or fibronectin.
14. The method of claim 13 , wherein the matrix is retronectin.
15. The method of claim 11 , wherein the defined media comprises ascorbic acid and/or nicotinamide.
16. The method of claim 15 , wherein the ascorbic acid is present at a concentration of 50 μM to 1 mM.
17. The method of claim 15 , wherein the nicotinamide is present at a concentration of 0.1 mM to 5 mM.
18. The method of claim 11 , wherein the Notch ligand is DLL4.
19. The method of claim 11 , wherein the one or more cytokines are selected from the group consisting of SCF, TPO, IL-7, and Flt-3.
20. The method of claim 11 , wherein step (ii) is two to four weeks.
21. The method of claim 11 , wherein the lymphoid cells express one or more of the markers selected from the group consisting of CD8, CD7, CD45, CD5, CD4 and CD3.
22. The method of claim 21 , wherein more than 5% of the lymphoid cells are positive for at least two of the markers.
23. The method of claim 21 , wherein more than 50% of the lymphoid cells are positive for CD3.
24. The method of claim 23 , wherein more than 60% of the lymphoid cells are positive for CD3.
25. The method of claim 11 , wherein the one or more cytokines comprise IL-2.
26. The method of claim 11 , wherein the one or more cytokines do not comprise IL-2.
27. The method of claim 10 , wherein the population of cells with increased lymphoid potential have an increase in fold enrichment of T cell generation as compared to an unsorted cell population or a population of cells negative for two more of the cell surface markers selected from the group consisting of CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4.