Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages
Transcripción
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.
