Method for generating T-cell progenitors
The invention relates to the field of cell therapy and to an in vitro method for generating T-cell progenitors, comprising the step of exposing CD34+ cells in a medium containing a Notch ligand, a soluble domain of the Delta-like ligand 4, joined to an Fc region of a protein IgG, in the presence of a fragment of fibronectin comprising the motifs RGDS and CS-1 and a heparin-binding domain.
1. An in vitro method of differentiating human cells that express CD34 into T-cell progenitors that express CD7 and optionally CD34, the method comprising culturing human cells that express CD34 for a period of between 3 to 8 days in the presence of:
(i) a medium,
(ii) a Notch ligand immobilized on a support, wherein the Notch ligand comprises a soluble domain of a Delta-like-4 ligand, fused to an Fc region of an IgG protein, and
(iii) a fibronectin fragment comprising an RGDS motif and/or a CS-1 motif,
such that T-cell progenitors that express CD7 and optionally CD34 are obtained.
2. The method process of claim 1 , wherein said IgG protein is an IgG2 protein.
3. The method process of claim 1 , wherein said fibronectin fragment is fragment CH-296 of human fibronectin.
4. The method of claim 1 , wherein said fibronectin fragment is immobilized on said support or on at least one other support.
5. The method of claim 1 , wherein said medium comprises a viral vector for at least a portion of the culturing period.
6. The method of claim 5 , wherein the viral vector is a lentiviral vector.
7. The method of claim 5 , wherein the viral vector is introduced in the medium after a period of preactivation of the CD34+ cells of between 4 hours and 36 hours.
8. The method of claim 5 , wherein the T cell progenitors expressing CD7 and optionally CD34 are for use in a method for treating a patient, and wherein the viral vector encodes a protein that is absent or deficient in the patient.
9. The method of claim 1 , wherein the medium comprises at least 15% of FBS.
10. The method of claim 1 , wherein the medium comprises at least three cytokines selected from the group consisting of interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO) and the Flt3 ligand (FLT3L).
11. The method of claim 10 , wherein the medium comprises IL-7, SCF, TPO and FLT3L.
12. The method of claim 1 , further comprising purifying the T-cell progenitors.
13. The method of claim 12 , further comprising packaging said T-cell progenitors in a bag.
14. The method of claim 1 , wherein the fibronectin fragment comprises a heparin-binding domain.
15. The method of claim 1 , wherein the fibronectin fragment comprises an RGDS motif, a CS-1 motif, and a heparin-binding domain.
16. The method of claim 1 , wherein said fibronectin fragment is immobilized on beads.