Microglia-sufficient brain organoids

A microglia-sufficient brain organoid comprising functionally active microglia cells, wherein the microglia-sufficient brain organoid is obtained by a step of incubating primitive-like macrophage cells with a brain organoid that is between 23 to 29 days old in cerebral organoid medium comprising CSF-1 in a low attachment cell culture vessel to generate functionally active microglia cells, wherein:

• a) the primitive-like macrophage cells are generated from a first population of stem cells by:
• i) incubating the stem cells in a culture medium comprising a GSK3 inhibitor, BMP4, and VEGF to differentiate the stem cells into cells of a mesoderm lineage;
• ii) incubating cells of the mesoderm lineage in a culture medium comprising FGF-2 to differentiate the cells of the mesoderm lineage into hemangioblast cells;
• iii) incubating the hemangioblast cells in a culture medium comprising VEGF and FGF-2;
• iv) incubating the hemangioblast cells further in a second culture medium comprising DKK1, SCF, FGF2, IL3, and IL6 to differentiate the hemangioblast cells to hematopoietic cells;
• v) incubating the hematopoietic cells in a culture medium comprising SCE, FGF-2, IL-3, and IL-6 to induce maturation of the hematopoietic cells to mature hematopoietic cells;
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• vi) incubating the mature hematopoietic cells in a culture medium comprising CSF-1 to differentiate the mature hematopoietic cells to primitive-like macrophage cells;
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• b) the brain organoid is generated from a second population of stem cells over a period of between 23 to 29 days by:
• i) incubating the second population of stem cells in a low attachment cell culture vessel to form an embryoid body;
• ii) incubating the embryoid body in neural induction medium to differentiate the embryoid body into an organoid comprising neuroectoderm cells;
• iii) embedding the organoid comprising neuroectoderm cells in Matrigel and incubating the organoid comprising neuroectoderm cells in cerebral organoid medium containing N2 and B27 without vitamin A to differentiate the organoid comprising neuroectoderm cells to an organoid comprising neural epithelial cells;
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• iv) incubating the organoid comprising neural epithelial cells with cerebral organoid medium containing N2 and B27 without vitamin A to differentiate the organoid comprising neural epithelial cells to the brain organoid.