Methods for immortalizing cells
The present invention provides methods for immortalizing precursor cells that are non-terminally differentiated cells such as stem cells, said methods comprising culturing the precursor cells in the presence of a Notch agonist and one or more growth factors that support the proliferation but not differentiation of the non-terminally differentiated cells. The present invention further provides methods to induce the differentiation of immortalized cells, comprising growing the cells in the presence of a Notch agonist and at least one growth factor which supports the differentiation of the cell into a more specialized cell type. The immortalized and/or differentiated cells of the invention can be used to repopulate cell populations that have been diminished, for example as a result of infection or exposure to certain drugs. The invention further provides a cell culture comprising a population of non-terminally differentiated cells immortalized by the methods of the present invention and kits comprising reagents that promote the immortalization of precursor cells. The invention further provides methods for screening for Notch modulators and for identifying genes involved in processes of cellular differentiation.
1. A kit comprising, in one or more containers,
(a) immobilized fibronectin or a fragment thereof;
(b) an immobilized Notch agonist comprising an extracellular, Notch-interacting domain of a Delta, a Jagged, or a Serrate protein; and
(c) Stem cell factor (SCF) in combination with two or more of Flt-3 ligand (Flt-3), Interleukin-6 (IL-6), Interleukin-3 (IL-3), Interleukin-11 (IL-11), thrombopoietin (TPO), Granulocyte-macrophage colony stimulating factor (GM-CSF), or granulocyte colony stimulating factor (G-CSF);
wherein said combination of (a), (b), and (c) are together effective to maintain a proliferating hematopoietic stem cell population comprising a majority of cells that do not terminally differentiate for a time period beyond which hematopoietic stem cells not in the presence of (a), (b), and (c) stop proliferating and/or die while exposed to them in culture.
2. The kit of claim 1 , further comprising in a separate container one or more purified growth factors that promote the differentiation of said hematopoietic stem cells.
3. The kit of claim 1 , wherein the Notch agonist comprises an extracellular, Notch-interacting domain of the Delta protein.
4. The kit of claim 3 , wherein the Notch agonist is in dimeric form.
5. The kit of claim 4 , wherein the Notch agonist in dimeric form is Delta ext-IgG .
6. The kit of claim 1 , wherein the fragment of fibronectin is CH-296.
7. The kit of claim 1 , wherein the two or more growth factors in combination with SCF are IL-6, IL-11, or Flt-3L.
8. The kit of claim 1 , wherein the two or more growth factors in combination with SCF are Flt-3L, TPO, IL-6, or IL-3.