Compositions and methods for enhanced generation of hematopoietic stem/progenitor cells
The present invention relates to methods, kits and compositions for expansion of hematopoietic stem/progenitor cells and providing hematopoietic function to human patients in need thereof. In one aspect, it relates to kits and compositions comprising a Notch agonist and an aryl hydrocarbon receptor antagonist. Also provided herein are methods for expanding the hematopoietic stem/progenitor cells using kits and compositions comprising a Notch agonist and an aryl hydrocarbon receptor antagonist. The hematopoietic stem/progenitor cells expanded using the disclosed kits, compositions and methods include human umbilical cord blood stem/progenitor cells, placental cord blood stem/progenitor cells and peripheral blood stem cells. The present invention also relates to administering hematopoietic stem/progenitor cells expanded using a combination of a Notch agonist and an aryl hydrocarbon receptor antagonist to a patient for short-term and/or long-term in vivo repopulation benefits.
1. A method of expanding hematopoietic stem/progenitor cells with improved in vivo repopulating ability, comprising culturing isolated hematopoietic stem/progenitor cells ex vivo in the presence of a composition comprising an amount of Delta ext-IgG and an amount of 4-(2-(2-(benzothiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (SR1) that results in expansion of hematopoietic stem/progenitor cell sample with improved in vivo repopulating ability as compared to a hematopoietic stem/progenitor cell sample expanded in culture comprising an amount of Delta ext-IgG but no SR1 or an amount of SR1 but no Delta ext-IgG , wherein the amount of SR1 is between about 200 nM and 1000 nM and wherein the amount of Delta ext-IgG is between about 1.25 μg/ml and 10 μg/ml, thereby expanding hematopoietic stem/progenitor cells with improved in vivo repopulating ability.
2. The method of claim 1 , wherein the isolated hematopoietic stem/progenitor cells are human.
3. The method of claim 1 , wherein in said culturing step, hematopoietic stem/progenitor cells are in contact with a solid phase on which the Delta ext-IgG is immobilized, and the SR1 is in a fluid contacting said cells.
4. The method of claim 1 , wherein in said culturing step, hematopoietic stem/progenitor cells are cultured on a solid phase coated with the Delta ext-IgG , and the SR1 is in a fluid contacting said cells.
5. The method of 1 , wherein the composition or the fluid further comprises one or more growth factors.
6. The method of claim 5 , wherein the one or more growth factors are selected from stem cell factor (SCF), 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), and granulocyte colony stimulating factor (G-CSF).
7. The method of claim 6 , wherein the composition or the fluid comprises IL-6, Flt-3, SCF and TPO.
8. The method of claim 3 , wherein the Delta ext-IgG is applied to the solid phase.
9. The method of claim 3 , wherein the amount of Delta ext-IgG is between about 2 and 6 μg/ml applied to the solid phase.
10. The method of claim 1 , wherein the composition further comprises an immobilized fibronectin or a fragment thereof.
11. The method of claim 10 , wherein the fragment of fibronectin is CH-296.
12. The method of claim 1 , wherein the isolated hematopoietic stem/progenitor cells are derived from umbilical cord blood and/or placental cord blood.
13. The method of claim 12 , wherein the isolated hematopoietic stem/progenitor cells are derived from a pool of two or more different humans at birth.
14. The method of claim 13 , wherein the isolated hematopoietic stem/progenitor cells are derived from humans of the same race.
15. The method of claim 13 , wherein the isolated hematopoietic stem/progenitor cells are derived from humans of the same ethnicity.
16. The method of claim 1 , wherein the percentage of CD34 + cells in the expanded hematopoietic stem/progenitor cell sample is higher than the percentage of CD34 + cells in the isolated hematopoietic stem/progenitor cells.
17. The method of claim 1 , wherein the percentage of CD34 + cells in the expanded hematopoietic stem/progenitor cell sample is higher than the percentage of CD34 + cells in a sample of the hematopoietic stem/progenitor cells expanded using Delta ext-IgG alone and/or SR1 alone.
18. The method of claim 1 , wherein the percentage of CD34 + CD90 + cells in the expanded hematopoietic stem/progenitor cell sample is higher than the percentage of CD34 + CD90 + cells in a sample of the hematopoietic stem/progenitor cells expanded using Delta ext-IgG alone and/or SR1 alone.
19. The method of claim 1 , wherein the percentage of CD34 − CD14 + cells in the expanded hematopoietic stem/progenitor cell sample is lower than the percentage of CD34 − CD14 + cells in the isolated hematopoietic stem/progenitor cells.
20. The method of claim 1 , wherein the percentage of CD34 − CD14 + cells in the expanded hematopoietic stem/progenitor cell sample is lower than the percentage of CD34 − CD14 + cells in a sample of the hematopoietic stem/progenitor cells expanded using Delta ext-IgG alone and/or SR1 alone.
21. A method of expanding human hematopoietic stem/progenitor cells with improved in vivo repopulating ability, comprising culturing isolated hematopoietic stem/progenitor cells ex vivo on a solid phase coated with (i) an amount of Delta ext-IgG and (ii) CH-296, and in the presence of a medium comprising (iii) an amount of an aryl hydrocarbon receptor antagonist and (iv) four or more growth factors;
wherein (i) and (iii) are provided in amounts that result in expanded human hematopoietic stem/progenitor cells with improved in vivo repopulating ability as compared to a hematopoietic stem/progenitor cell sample expanded in culture comprising an amount of Delta ext-IgG but no aryl hydrocarbon receptor antagonist or an amount of an aryl hydrocarbon receptor antagonist but no Delta ext-IgG ; and
wherein the aryl hydrocarbon antagonist is 4-(2-(2-(benzothiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; and wherein the four or more growth factors are selected from IL6, TPO, Flt-3 ligand, CSF and IL3;
thereby producing an expanded hematopoietic stem/progenitor cell sample with improved in vivo repopulating ability.
22. The method of claim 1 , wherein the Delta ext-IgG is Delta ext-IgG .