Differentiation of macrophages from pluripotent stem cells

A method for producing cells that express a microglial marker from human pluripotent stem cells, wherein the microglial marker is IBA1, the method comprising:

• (i) generating primitive macrophages from said human stem cells, wherein the primitive macrophages are generated by:
• (a) contacting and incubating said stem cells with a serum-free culture media comprising VEGF, BMP4 and a GSK3 inhibitor under normoxic conditions such that said stem cells differentiate into cells of the mesoderm lineage;
• (b) contacting and incubating said cells of the mesoderm lineage under hypoxic conditions with a culture media comprising SCF, FGF-2, IL-3, IL-6, VEGF and DKK1 such that the cells of the mesoderm lineage differentiate into cells of the hematopoietic cell lineage;
• (c) culturing said cells of the hematopoietic cell lineage with a culture media comprising SCF, FGF-2, IL-3 and IL-6 under normoxic conditions such that the cells of the hematopoietic cell lineage mature;
• (d) contacting and incubating said mature cells of the hematopoietic cell lineage with a culture media comprising M-CSF under normoxic conditions such that said hematopoietic cells differentiate into primitive macrophages that express CD163, CD11b, HLA-DR, IBA-1, CX3CR1 and CD14;
• (ii) generating neurons from said human stem cells, wherein the neurons are generated by:
• (e) contacting and incubating said stem cells with neural progenitor cell (NPC) medium for up to 7 days such that said stem cells differentiate to neural precursor cells;
• (f) contacting and incubating the neural precursor cells from operation (e) with a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor;
• (g) contacting and incubating the cells from operation (f) with NPC media comprising epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF);
• (h) contacting and incubating the cells from operation (g) with neuronal differentiation media such that the cells from operation (g) generate neurons;
• and
• (iii) contacting and incubating the primitive macrophages from (i) with the neurons from (ii) for up to 21 days such that the neurons from (ii) generate the cells expressing the microglial marker.

A cell expressing a microglial marker IBA1 obtained by a method comprising:

• (i) generating primitive macrophages from said human stem cells, wherein the primitive macrophages are generated by:
• (a) contacting and incubating said stem cells with a serum-free culture media comprising VEGF, BMP4 and a GSK3 inhibitor under normoxic conditions such that said stem cells differentiate into cells of the mesoderm lineage;
• (b) contacting and incubating said cells of the mesoderm lineage under hypoxic conditions with a culture media comprising SCF, FGF-2, IL-3, IL-6, VEGF and DKK1 such that the cells of the mesoderm lineage differentiate into cells of the hematopoietic cell lineage;
• (c) culturing said cells of the hematopoietic cell lineage with a culture media comprising SCF, FGF-2, IL-3 and IL-6 under normoxic conditions such that the cells of the hematopoietic cell lineage mature;
• (d) contacting and incubating said mature cells of the hematopoietic cell lineage with a culture media comprising M-CSF under normoxic conditions such that said hematopoietic cells differentiate into primitive macrophages that express CD163, CD11b, HLA-DR, IBA-1, CX3CR1 and CD14;
• (ii) generating neurons from said human stem cells, wherein the neurons are generated by:
• (e) contacting and incubating said stem cells with neural progenitor cell (NPC) medium for up to 7 days such that said stem cells differentiate to neural precursor cells;
• (f) contacting and incubating the neural precursor cells from operation (e) with a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor;
• (g) contacting and incubating the cells from operation (f) with NPC media comprising epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF);
• (h) contacting and incubating the cells from operation (g) with neuronal differentiation media such that the cells from operation (g) generate neurons;
• and
• (iii) contacting and incubating the primitive macrophages from (i) with the neurons from (ii) for up to 21 days such that the neurons from (ii) generate the cells expressing the microglial marker.