This article outlines a protocol for differentiating human-induced pluripotent stem cells into microglia-like cells using embryoid bodies. The process involves several steps including cell aggregation, differentiation into primitive macrophage precursors, and subsequent maturation into microglia-like cells.
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.
For plating cells, add 100 microliters of the diluted cells per well into a low-adherence round-bottom 96-well plate. Centrifuge the plate at 125 times g for three minutes, and incubate at 37 degrees Celsius and 5% carbon dioxide for four days.
On day 2, using a multichannel pipette, replace the old half EB medium with a fresh medium. To generate primitive macrophage precursors or PMPs, coat the wells of a 6-well plate by adding 1 milliliter of ice-cold matrigel coating solution. Then, incubate the plate at 37 degrees Celsius and 5% carbon dioxide for two hours or overnight.
On day 4 of EB differentiation, using a 1-milliliter pipette tip, transfer the EBs to the matrigel-coated wells. Pipette up and down to dislodge the EBs from the well. Then, hold the plate tilted to allow the EBs to settle down at the edge of the well.
Once all the EBs have settled down, gently pipette and replace the old medium while keeping the EBs at the edge of the well with 3 milliliters of freshly prepared PMP complete medium. Evenly distribute the cells in the wells by manually shuffling the plates side-to-side and back-to-front. Then, incubate the plate for seven days to allow the EBs to attach to the bottom of the well.
After seven days, inspect the EBs under a light field microscope at 4 times magnification to ensure that they are attached to the bottom of the wells. Perform a half medium change, and on day 21, replace the complete medium with 3 milliliters of PMP complete medium.
On day 28, look for round cells referred to as PMPs in the supernatant. Then, collect the medium containing the PMPs using a 10-milliliter pipette and automatic pipettor without disturbing the EBs.
Transfer the PMPs in the medium to a 15-milliliter conical tube. Centrifuge the collected PMPs at 200 times g for four minutes and aspirate the supernatant before resuspending them in 1 to 2 milliliters of microglia-like cells or IMG basal medium.
Then, count the cells on the hemocytometer, as described previously. Centrifuge the rest of the cells and dilute the PMPs to the desired concentration. Then, plate the cells at a density of 105 cells per square centimeter on cell culture-treated plates using freshly prepared IMG complete medium.
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