Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

Adult somatic cells can be genetically reprogrammed to obtain induced pluripotent stem cells, iPSCs, that exhibit stem cell-like undifferentiated and proliferative states.

To differentiate human iPSCs into macrophages — white blood cells that phagocytose pathogens and cellular debris — begin with a culture plate with a non-cell adhesive surface.

Add media containing the growth factors bone morphogenetic protein, stem cell factor, and vascular endothelial growth factor. Seed the iPSCs and incubate.

The non-adhesive surface prevents iPSC attachment and maintains the cells in suspension, facilitating aggregation into three-dimensional embryoid bodies, EBs. The growth factors initiate iPSC differentiation within the EBs into mesodermal lineage cells.

Transfer the differentiated EBs to a centrifuge tube. Allow them to settle through gravity. Resuspend the EBs in fresh media containing interleukin-3, IL3, and macrophage colony-stimulating factor, CSF1. Plate the EBs on a gelatin-coated cell culture plate.

Gelatin — an extracellular matrix protein — facilitates EB attachment. IL3 stimulates the EBs to differentiate into hematopoietic stem-progenitor cells, giving rise to myeloid cells. CSF1 supports myeloid cell proliferation and their differentiation into macrophage precursors that get released and remain in suspension in the media.

Collect the suspension cells and centrifuge. Resuspend the cells in CSF1-supplemented media.

Plate the cells on uncoated culture plates.

CSF1 induces terminal differentiation of the macrophage precursors into fully-functional, adherent mature macrophages.

To begin the differentiation process on day zero, add 2.25 milliliters of stage 1 media into two wells of an ultra-low attachment 6-well plate. Then, for 180% confluent well of iPSCs in a 6-well plate, replace the maintenance media with 1.5 milliliters of stage 1 media. Cut colonies using a new cell passaging tool. Using a pipette, transfer the cut colonies into the two wells of the ultra-low attachment 6-well plate.

On day 2, adjust the cytokine concentrations by adding 0.5 milliliters of hESC-SFM media to the 6-well well plate containing the day two embryoid bodies.

On day 4, coat four wells of a 6-well tissue culture plate with 0.1% gelatin. After incubating for at least 10 minutes, remove the gelatin, and add 2.5 milliliters of stage 2 media.

Collect formed embryoid bodies from the two wells of a 6-well plate, and place them in a 50-milliliter centrifuge tube. Allow them to settle at the bottom of the tube by gravity. Then, carefully aspirate the media from the tube, and resuspend the embryoid bodies in 2 milliliters of stage 2 media.

Transfer 10 to 15 embryoid bodies to a gelatin-coated well, containing 2.5 milliliters of stage 2 media, and incubate at 37 degrees Celsius with 5% carbon dioxide. For two to three weeks, change the media every three to four days.

Plating of the EBs is crucial. Having fewer than 10 or more than 15 EBs, or having them unevenly distributed, can lead to low yields of macrophages.

After two to three weeks, the embryoid bodies start releasing non-adherent hematopoietic cells into suspension. Using a 40-micrometer strainer, collect these cells into a 50-milliliter tube, and then, replenish the media. Centrifuge the suspension of hematopoietic cells at 200 x g for three minutes.

Resuspend the hematopoietic cells in stage 3 media. Then, plate the cells at a density of 0.2 x 10⁶ per milliliter. Change the stage 3 media every five days.

After nine to 11 days the macrophages will be fully differentiated and fully functional.