Method for generating human dendritic cells for immunotherapy
In various embodiments methods of producing a cell population enriched for CLEC9A+ dendritic cells are provided where the methods involve culturing stem cells and/or progenitor cells in a cell culture comprising culture medium, a notch ligand, stem cell factor (SCF), FLT3 ligand (FLT3L); thrombopoietin (TPO); and IL-3 and/or GMCSF.
1. A method of producing a cell population enriched for CLEC9A+ dendritic cells in a cell culture comprising culture medium, said method comprising co-culturing stem cells and/or progenitor cells with a human or murine stromal cell line transduced or transfected with a nucleic acid that encodes and expresses a notch ligand, wherein the notch ligand is DLL4, DLL1, Jagged 1 (JAG1), or Jagged 2 (JAG2), or a Notch-binding fragment of DLL4, DLL1, Jagged 1 (JAG1), or Jagged 2 (JAG2), wherein said culture medium is supplemented with L-alanyl-L-glutamine dipeptide.
2. The method of claim 1 , wherein said cell culture comprises one or more of stem cell factor (SCF), FLT3 ligand (FLT3L), thrombopoietin (TPO), IL-3 and GM-CSF.
3. The method of claim 2 , wherein said cell culture comprises: SCF, FLT3L, and IL-3 or GM-CSF.
4. The method of claim 1 , wherein said stem cells and/or progenitor cells comprise hematopoietic stem cell and/or progenitor cells (HSPCs), and wherein said cells are enriched for CD34+ cells.
5. The method of claim 1 , wherein said stem cells or progenitor cells are derived from bone marrow, umbilical cord, peripheral blood, or mobilized peripheral blood.
6. The method of claim 1 , wherein said stem cells and/or progenitor cells comprise embryonic stem cells, adult stem cells, or induced pluripotent stem cells.
7. The method of claim 1 , wherein said notch ligand is provided by co-culture with stroma cells comprising stem cells.
8. The method of claim 7 , wherein said stem cells are mesenchymal stem cells (MSCs).
9. The method of claim 7 , wherein said stroma cells comprise induced pluripotent stem cells (IPSCs) or derivatives of IPSCs, or human embryonic stem cells.
10. The method of claim 1 , wherein said culture comprises :minimum essential medium (MEM) with L-alanyl-L-glutamine dipeptide; fetal calf serum or human AB serum; recombinant SCF; recombinant FLT3L; and IL-3 or GM-CSF.
11. The method of claim 10 , wherein said culture comprises:
about 5% human AB serum;
about 5 ng/ml SCF;
about 5 ng/ml FLT3L; and
about 5 ng/ml IL-3 or about 10 ng/ml GM-CSF.