Haematopoietic stem/progenitor cells

Abstract

The invention relates to an isolated cell having, after culturing a pluripotent stem cell (PSC) in a medium comprising a WNT agonist and an ACTIVIN antagonist, increased HOXA gene expression relative to a PSC not cultured in a medium comprising a WNT agonist and an ACTIVIN antagonist, wherein the cell with increased HOXA gene expression is capable of generating a definitive haematopoietic stem/precursor cell. The invention also relates to use of the cell for generating a definitive haematopoietic stem/precursor cell, and a method for differentiating a PSC into a definitive haematopoietic stem/precursor cell and a definitive haematopoietic stem/progenitor cell differentiated from a PSC by the method. The invention further relates to a therapeutic composition comprising a cell of the invention, and to therapeutic methods and uses of a cell of the invention. The invention also relates to a reporter cell comprising distinguishable SOX 7 and RUNXIC reporters, for use in tracking the differentiation of a PSC into a definitive haematopoietic stem/progenitor cell.

1 .- 8 . (canceled) 
     
     
         9 . A method for differentiating a PSC into a definitive haematopoietic stem/progenitor cell, the method comprising increasing HOXA gene expression by culturing the PSC in a medium comprising a WNT agonist and an ACTIVIN antagonist, wherein increased HOXA gene expression is relative to a PSC not cultured in a medium comprising a WNT agonist and an ACTIVIN antagonist. 
     
     
         10 . The method of  claim 9 , wherein the HOXA gene is one or more of HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXA11, and HOXA13. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 9 , wherein culturing the PSC in a medium comprising a WNT agonist and an ACTIVIN antagonist comprises culturing for about 2 days. 
     
     
         13 . The method of  claim 12 , wherein culturing the PSC in a medium comprising a WNT agonist and an ACTIVIN antagonist comprises adding the WNT agonist and the ACTIVIN antagonist to the medium at about day 3. 
     
     
         14 . The method of  claim 9 , wherein the WNT agonist is CHIR99021, optionally wherein the medium comprises 3 μM CHIR99021. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 9 , wherein the ACTIVIN antagonist is SB431542, optionally wherein the medium comprises 3-4 μM SB431542. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 9 , wherein the medium further comprises bone morphogenic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), ACTIVIN A and fibroblast growth factor 2 (FGF2), and culturing is from about day 0 to about day 4, optionally wherein the medium comprises about 20-ng/mL BMP4, about 20-30 ng/mL VEGF, about 40 ng/mL SCF, about 10-20 ng/nL ACTIVIN A, and about 10 ng/mL FGF2. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 9 , wherein the medium further comprises a retinoid or retinoic acid analogue from day 2, day 3, day 4 or day 5. 
     
     
         21 . The method of  claim 9 , wherein the PSC is cultured as an embryoid body. 
     
     
         22 . The method of  claim 9 , further comprising replacing the medium with medium comprising BMP4, VEGF, SCF, FGF2 and insulin-like growth factor 2 (IGF2) at about day 5, optionally wherein the medium comprises about 20 ng/mL BMP4, about 50 ng/mL VEGF, about 50 ng/mL SCF, about 10 ng/mL FGF2, and about 30 ng/mL IGF2. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 22 , comprising culturing the PSC for about 2 days. 
     
     
         25 . The method of  claim 9 , further comprising transferring the PSC to a culture surface comprising an extracellular matrix protein at about day 7 and culturing the PSC adherently in medium comprising VEGF, SCF, FGF2, IGF2, interleukin 6 (IL-6), thrombopoietin (TPO), FLT3 receptor ligand (FLT3L), and optionally interleukin 3 (IL-3) and/or erythropoietin (EPO), optionally wherein the medium comprises about 50 ng/mL VEGF, about 50 ng/mL SCF, about 25 ng/mL IL-6, about 30 ng/mL TPO, about ng/mL FLT3L, about 3 U/mL EPO, about 10 ng/mL FGF2, about 50 ng/mL SCF and about ng/mL IGF2, and optionally about 25-30 ng/mL IL-3 and/or about 3 U/mL EPO. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 9 , wherein the medium is APEL medium. 
     
     
         28 . The method of  claim 9 , wherein the definitive haematopoietic stem/progenitor cell is capable of producing a SOX17 +  blood vessel cell. 
     
     
         29 . The method of  claim 9 , wherein the definitive haematopoietic stem/progenitor cell is capable of producing a RUNX1C +  blood cell. 
     
     
         30 . The method of  claim 9 , wherein the PSC is an embryonic stem cell (ESC) or an induced PSC (iPSC). 
     
     
         31 . The method of  claim 9 , wherein the PSC is human. 
     
     
         32 . A definitive haematopoietic stem/progenitor cell differentiated from a PSC by the method of  claim 9 . 
     
     
         33 .- 34 . (canceled) 
     
     
         35 . A method for treating a condition, disease or disorder requiring HSC transplantation, the method comprising administering to a subject a definitive haematopoietic stem/progenitor cell differentiated from a PSC by the method of  claim 9 . 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein the condition, disease or disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (relapsed, refractory), Non-Hodgkin (relapsed or refractory) lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, a myelodysplastic syndrome, a glioma, other solid tumour, thalassemia, sickle cell anemia, aplastic anemia, Fanconi anemia, an immune deficiency syndrome, or an inborn error of metabolism.