Method of differentiation of pluripotent stem cells to hematopoietic precursor and stem cells

A method of producing natural killer (NK) cells comprising:

• a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and/or a bone morphogenetic protein (BMP), wherein the PSCs are grown on a substrate for about 1-8 days;
• b) contacting the culture of PSCs with a vascular endothelial growth factor (VEGF) alone or in combination with a WNT signaling pathway activator and/or a BMP for about 1-8 days following step a), thereby generating a population of CD34+ precursor cells, wherein the cells produced after step b) are at least about 80% enriched for CD34+ cells in the total population of cells;
• and
• c) contacting the population of CD34+ precursor cells with one or more of interleukin-7 (IL-7), IL-15, SCF and FMS-like tyrosine kinase 3 ligand (FLT3L),
• thereby producing NK cells.

A method of inducing natural killer (NK) cell differentiation from pluripotent stem cells (PSCs) comprising:

• a) generating CD34+ hemogenic endothelium (HE) cells by:
• (i) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP) for about 3 days;
• and
• (ii) contacting the adherent culture of PSCs of i) with a vascular endothelial growth factor (VEGF) for about 4 days:
• thereby generating a population of cells comprising at least 80% CD34+ HE cells;
• b) contacting the CD34+ HE cells of a) with one or more of IL-7, IL-15, SCF and/or FLT3L for about 7 days, thereby generating a transient population of cells comprising at least 80% CD34+/CD45+ hematopoietic stem cells (HSCs);
• and
• c) subsequently contacting the CD34+/CD45+ HSCs of b) with one or more of IL-7, IL-15, FLT3L and SCF for at least about 7-21 days,
• thereby inducing NK cell differentiation from PSCs.

A kit comprising:

• a) an hemogenic endothelium (HE) induction cocktail comprising:
• a WNT signaling pathway activator, a bone morphogenetic protein (BMP) and/or a vascular endothelial growth factor (VEGF)
• b) a natural killer (NK) induction cocktail comprising interleukin-7 (IL-7), IL-15, SCF and/or FLT3L;
• and
• c) instructions for inducing pluripotent stem cell (PSC) differentiation into NK cells.

A method of generating terminally differentiated hematopoietic cells from pluripotent stem cells (PSCs) comprising:

• a) generating CD34+ hematopoietic precursor cells by:
• (i) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs are grown on a substrate for about 2-5 days;
• and
• (ii) contacting the adherent culture of PSCs with a vascular endothelial growth factor (VEGF) for about 2-5 days following step (i), wherein the cells produced after step (ii) are at least about 80% enriched for CD34+ cells in the total population of cells;
• thereby generating CD34+ precursor cells;
• and
• b) contacting the CD34+ precursor cells of a) with a mixture of agents to induce differentiation of the CD34+ precursor cells into terminally differentiated hematopoietic cells, thereby generating terminally differentiated hematopoietic cells.

A method of producing a population of CD34+ hematopoietic stem cells comprising:

• a) contacting a culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs are grown on a substrate for about 1-8 days;
• and
• b) contacting the culture of PSCs with a vascular endothelial growth factor (VEGF) alone or in combination with a WNT signaling pathway activator and/or a BMP for about 1-8 days following step a),
• wherein the cells produced after step b) are at least about 80% enriched for CD34+ hematopoietic stem cells in the total population of cells,
• thereby producing a population of CD34+ hematopoietic stem cells.

A method of generating hematopoietic stem cells from pluripotent stem cells (PSCs) comprising:

• a) contacting an adherent culture of induced pluripotent stem cells (iPSCs) with a WNT signaling pathway activator and a bone morphogenetic protein (BMP), wherein the PSCs are grown on a substrate for about 2-5 days;
• and
• b) contacting the adherent culture of PSCs with a vascular endothelial growth factor (VEGF) for about 2-5 days following step (a), wherein the cells produced after step (b) are at least about 80% enriched for CD34+ cells in the total population of cells;
• thereby generating CD34+ precursor cells,
• thereby generating hematopoietic stem cells.