Expansion and engraftment of stem cells using Notch 1 and/or Notch 2 agonists
The present disclosure provides methods for immortalizing precursor cells that are non-terminally differentiated cells such as stem cells, the methods comprising culturing the precursor cells in the presence of a Notch 1 agonist, Notch 2 agonist or Notch 1 agonist and Notch 2 agonist (and, in particular embodiments, one or more growth factors) that support the proliferation but not differentiation of the non-terminally differentiated cells. The present disclosure further provides methods to induce the differentiation of immortalized cells, comprising growing the cells in the presence of a Notch 1 agonist, Notch 2 agonist or Notch 1 agonist and Notch 2 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 disclosure can be used to repopulate cell populations that have been diminished, for example as a result of infection or exposure to certain drugs. The disclosure further provides a cell culture comprising a population of non-terminally differentiated cells immortalized by the methods of the present disclosure and kits comprising reagents that promote the immortalization of precursor cells.
1. A method for expanding hematopoietic stem/progenitor cells, comprising culturing the hematopoietic stem/progenitor cells in the presence of
(a) an immobilized Notch 2 agonist which is an antibody or an antigen-binding fragment thereof that specifically binds to Notch 2 within an extracellular EGF repeat domain and exhibits substantially no binding to Notch 1, and
(b) growth factors,
thereby producing an expanded hematopoietic stem/progenitor cell population.
2. The method of claim 1 , further comprising detecting Notch 2 expression by the hematopoietic stem/progenitor cells before and/or during the culturing; and using an amount of the Notch 2 agonist in the culture based on the detecting.
3. The method of claim 1 , wherein the antigen-binding fragment thereof of the Notch 2 agonist is selected from an Fv, Fab, Fab′, F(ab′) 2 , or single chain Fv fragment (scFv).
4. The method of claim 1 , wherein the hematopoietic stem/progenitor cells are obtained from bone marrow, umbilical cord blood, placental blood, or Wharton's jelly.
5. The method of claim 1 , wherein the hematopoietic stem/progenitor cells are obtained from fetal or neonatal blood.
6. The method of claim 1 , wherein during the culturing the immobilized Notch 2 agonist is at a concentration of 0.1 μg/ml to 50 μg/ml.
7. The method of claim 1 , wherein the growth factors are interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3 ligand.
8. The method of claim 7 , wherein during the culturing IL-3 is at a concentration of 10 ng/ml.
9. The method of claim 7 , wherein during the culturing one or more of IL-3, IL-6, TPO, SCF, and Flt-3 ligand are at a concentration of 50 ng/ml.
10. The method of claim 1 , wherein the culturing is for a time period of seven days to six weeks.
11. The method of claim 1 , wherein the culturing is for a time period of at least five weeks.
12. The method of claim 1 , wherein the hematopoietic stem/progenitor cells are human and Notch 2 is human.
13. The method of claim 1 , wherein the growth factors are human growth factors.
14. The method of claim 1 , wherein the Notch 2 agonist is immobilized on a solid phase.
15. The method of claim 14 , wherein the solid phase is the surface of a tissue culture dish or flask or a bead.
16. The method of claim 1 , wherein the hematopoietic stem/progenitor cells are (i) hematopoietic stem cells, (ii) hematopoietic progenitor cells, or (iii) hematopoietic stem and progenitor cells.
17. The method of claim 1 , wherein the hematopoietic stem/progenitor cells are short-term marrow engrafting cells.
18. The method of claim 1 , wherein the method further comprises exposing the expanded hematopoietic stem/progenitor cell population to an amount of the Notch 2 agonist and/or growth factors that promote differentiation to produce early T cell stem/progenitor cells able to migrate to the thymus and generate mature T cells.
19. A method for expanding hematopoietic stem/progenitor cells, comprising culturing the hematopoietic stem/progenitor cells in the presence of
(a) an immobilized Notch 1 agonist which is an antibody or an antigen-binding fragment thereof that specifically binds to an extracellular EGF repeat domain of Notch 1, and
(b) growth factors,
thereby producing an expanded hematopoietic stem/progenitor cell population.