Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells
Transcription
Begin with a low-adherence multi-well plate containing human-induced pluripotent stem cells in an embryoid body or EB medium.
Centrifuge to settle the cells. Incubate to promote cell proliferation and aggregation, forming embryoid bodies.
The cell lineage regulators promote cell differentiation into myeloid progenitors.
Collect these EBs and transfer them to the edge of an extracellular matrix or ECM-coated plate.
Replace the medium with a primitive macrophage precursor or PMP differentiation medium and gently agitate. Incubate to facilitate the EBs' attachment to the ECM.
Replace half of the medium and incubate.
The hematopoietic growth factors drive myeloid progenitors to differentiate into primitive macrophage precursors, which separate from the EBs and appear in the medium.
Collect these PMPs and spin down the cells. Resuspend them in a microglia differentiation medium.
Seed these cells in a laminin-coated multi-well plate and incubate.
PMPs utilize the differentiation factors and nutrients, differentiating into microglia-like cells, a type of brain immune cell.
