Method for obtaining T cells from pluripotent stem cells, and uses thereof
A method for obtaining a population of T cells from pluripotent stem cells, which includes a first step of obtaining hematopoietic stem cells and a second step of obtaining T cells. Also, the cell populations thus obtained according to this method and these cell populations for use as a medicament.
1. A method for obtaining a population of T cells from pluripotent stem cells, comprising the following steps:
a) culturing pluripotent stem cells under conditions allowing to obtain embryoid bodies comprising at least 5% of CD34+CD43+cells;
b) dissociating said embryoid bodies;
c) culturing said dissociated embryoid bodies with at least one Notch ligand; and
d) carrying out, during step c), the introduction between day Dd8 and day Dd12 of step c) of a vector comprising at least one nucleic acid sequence encoding Foxp3 in at least one cell.
2. The method according to claim 1 , wherein step c) of culturing the dissociated embryoid bodies with at least one Notch ligand is carried out by coculturing the dissociated embryoid bodies with cells expressing at least one Notch ligand.
3. The method according to claim 1 , wherein step d) of introducing a vector is carried out when at least 15% of the cell population exhibits the CD43− phenotype.
4. The method according to claim 1 , wherein the embryoid bodies comprise at least 15% of CD34+CD43+cells.
5. The method according to claim 1 , wherein step a) is carried out for at least 9 days.
6. The method according to claim 1 , wherein step a) is carried out for 9 to 12 days.
7. The method according to claim 1 , wherein step a) is carried out in a serum free culture medium comprising BMP, FGF2, VEGF, SCF, Flt3-L and/or IL-3.
8. The method according to claim 1 , wherein the dissociated embryoid bodies, obtained at the end of step b), are cultured in a culture medium comprising SCF, Flt3-L and/or IL-7, for at least 15 days.
9. The method according to claim 1 , wherein the vector of step d) is a retrovirus.
10. The method according to claim 1 , wherein the vector of step d) is a lentivirus.
11. The method according to claim 1 , wherein the nucleic acid sequence of the vector of step d) comprises a further nucleic acid sequence of interest.
12. The method according to claim 11 , wherein the further nucleic acid of interest is selected from a nucleic acid encoding a chimeric antigen receptor (CAR), Mcl-1, Bcl -xL, and Helios.
13. The method according to claim 1 , wherein the pluripotent stem cells are human induced pluripotent stem cells or human embryonic stem cells.
14. The method according to claim 1 , comprising an additional step of isolating a cell population selected from regulatory T cells (Treg), effector T lymphocytes (Teff), and CD4−CD8−CD3+TCRab+T lymphocytes.