Microglia-sufficient brain organoids

A method for generating a microglia-sufficient brain organoid having functionally active microglia cells, comprising a step of incubating primitive-like macrophage cells with a brain organoid that is between 23 days old 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 said stem cells in a culture medium comprising a GSK3 inhibitor, BMP4, and VEGF to differentiate said stem cells into cells of a mesoderm lineage;
• ii) incubating said cells of the mesoderm lineage in a culture medium comprising FGF-2 to differentiate said cells of the mesoderm lineage into hemangioblast cells;
• iii) incubating said hemangioblast cells in a culture medium comprising VEGF and FGF-2;
• iv) incubating said hemangioblast cells in a culture medium comprising DKK1, SCF, FGF2, IL3, and IL6 to differentiate said hemangioblast cells to hematopoietic cells;
• v) incubating said hematopoietic cells in a culture medium comprising SCF, FGF-2, IL-3, and IL-6 to induce maturation of said hematopoietic cells;
• vi) incubating said matured hematopoietic cells in a culture medium comprising CSF-1 to differentiate said matured hematopoietic cells to primitive-like macrophage cells;
• and
• 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 said second population of stem cells in a low attachment cell culture vessel to form an embryoid body;
• ii) incubating said embryoid body in neural induction medium to differentiate said 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 said organoid comprising neuroectoderm cells to an organoid comprising neural epithelial cells;
• iv) incubating the organoid comprising neural epithelial cells with cerebral organoid medium containing N2 and B27 without vitamin A to differentiate said organoid comprising neural epithelial cells to said brain organoid.