Compositions and methods for expansion of embryonic hematopoietic stem cells
The present invention relates to methods, kits and compositions for expansion of embryonic hematopoietic stem cells and providing hematopoietic function to human patients in need thereof. In one aspect, it relates to kits and compositions comprising a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway. Also provided herein are methods for expanding embryonic hematopoietic stem cells using kits and compositions comprising a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway. The embryonic hematopoietic stem cells expanded using the disclosed kits, compositions and methods include cells derived from an embryo (e.g., aorta-gonad-mesonephros region of the embryo), embryonic stem cells, induced pluripotent stem cells, or reprogrammed cells of other types. The present invention also relates to administering the embryonic hematopoietic stem cells expanded using a combination of a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway to a patient for short-term and/or long-term in vivo repopulation benefits.
1. A method of expanding embryonic hematopoietic stem cells, comprising:
culturing the embryonic hematopoietic stem cells ex vivo in the presence of
(i) a composition comprising an immobilized Delta or Jagged Notch agonist; and
(ii) a fluid medium comprising an antibody that inhibits TGFβ and growth factors comprising stem cell factor (SCF), Flt-3 ligand (Flt-3L), Interleukin-3 (IL-3), thrombopoietin (TPO), Interleukin-11 (IL-11), and Interleukin-6 (IL-6);
thereby producing an expanded embryonic hematopoietic stem cell sample.
2. The method of claim 1 , wherein the culturing is not in the presence of an aryl hydrocarbon receptor antagonist.
3. The method of claim 1 , wherein the immobilized Delta Notch agonist is an extracellular domain of a Delta protein.
4. The method of claim 3 , wherein the extracellular domain is fused to an Fc region of an IgG.
5. The method of claim 3 , wherein the Delta protein is a human Delta protein.
6. The method of claim 1 , wherein the immobilized Notch agonist is Delta1 ext-IgG and wherein Delta1 ext-IgG has been applied to a solid phase at a concentration of about 2.5 μg/ml.
7. The method of claim 1 , wherein the immobilized Jagged Notch agonist is an extracellular domain of a Jagged protein.
8. The method of claim 1 , wherein the SCF and Flt-3L are each in the fluid medium at a concentration between about 10 and 200 ng/ml, and wherein the IL-6 is in the fluid medium at a concentration between about 10 and 100 ng/ml.
9. The method of claim 1 , wherein the composition further comprises fibronectin or a fragment thereof.
10. The method of claim 1 , wherein the composition further comprises CH-296, and wherein in said culturing step, CH-296 is immobilized on a solid phase.
11. The method of claim 1 , wherein the fluid medium further comprises fetal bovine serum (FBS).
12. The method of claim 1 , wherein the embryonic hematopoietic stem cells are derived from an embryo, from embryonic stem cells (ESC), from induced pluripotent stem cells (iPSC), or from reprogrammed non-pluripotent cells.
13. The method of claim 1 , wherein the embryonic hematopoietic stem cells are derived from induced pluripotent stem cells (iPSC).
14. The method of claim 1 , wherein the embryonic hematopoietic stem cells are derived from a single human or more than one human.
15. The method of claim 1 , wherein the embryonic hematopoietic stem cells cultured in said culturing step are derived from cells of a patient with a genetic disorder associated with a gene having a sequence defect.
16. The method of claim 1 , wherein the embryonic hematopoietic stem cells are derived from the aorta-gonad-mesonephros (AGM) region of an embryo.
17. The method of claim 1 , wherein the cells cultured in said culturing step are a population of cells enriched for CD45 and/or VE-Cadherin, in which said embryonic hematopoietic stem cells are present.
18. A method of expanding human embryonic hematopoietic stem cells, comprising culturing the human embryonic hematopoietic stem cells ex vivo on a solid phase coated with Delta1 ext-IgG and CH-296, and in the presence of a fluid medium comprising:
(i) an antibody that inhibits TGFβ; and
(ii) growth factors comprising SCF, Flt-3L, IL-3, TPO, IL-11, and IL-6; thereby producing an expanded embryonic hematopoietic stem cell sample.
19. The method of claim 18 , wherein the Delta1 ext-IgG comprises a human Delta1 fragment and a human IgG fragment, the CH-296 is a human fibronectin fragment, and the SCF, Flt-3L and IL-6 are all human.