Fig. 1

Principles of iMph differentiation used in different OP9-independent protocols. iPSC differentiation into iMphs goes on through four main stages: the induction of mesoderm and hemogenic endothelium (HE), the induction of hematopoietic differentiation, myeloid specification of hematopoietic progenitors and terminal differentiation of the generated monocyte-like cells into iMphs. In EB-S protocols, to induce mesoderm and HE, iPSCs are cultured in low-adhesive conditions, which stimulate the formation of 3D cell aggregates, embryoid bodies (EBs). Within the EBs, mesoderm and HE are generated spontaneously, due to the tight intercellular interactions. After EBs are formed, they are transferred to tissue culture (TC) plates and cultured in the presence of IL-3 and M-CSF that induce the formation of hematopoietic progenitors and their myeloid specification. When monocyte-like cells appear in the culture, they are transferred to new TC plates, where their terminal differentiation into iMphs is directed by M-CSF. The remaining cultures are restimulated with IL-3 and M-CSF to induce new rounds of myeloid cell generation. In EB-F protocols, mesoderm/HE are also induced through the formation of EBs. However, differently from EB-S protocols, exogenous factors are added to the cultures to support the mesodermal pathway of cell differentiation. This increases the reproducibility and the efficacy of the protocols. Subsequent stages are induced either by culturing EBs in the presence of IL-3 and M-CSF (like in EB-S protocols) or by adding mixes of exogenous factors, that sequentially drive the cells through the hematopoietic and myeloid differentiation stages. 2D-F protocols do not imply EB formation. iPSCs are cultured in TC plates, where complex mixes of exogenous factors are sequentially added to drive the cells through the differentiation process. Color clues: Blue, mesoderm/HE induction; Green, hematopoietic differentiation; Orange, myeloid specification; Green/orange shaded, hematopoietic and myeloid differentiations are induced simultaneously; Pink, iMph terminal differentiation. Asterisk, exogenous factors and other special conditions used at the indicated stages. BMP4 Bone Morphogenetic Protein 4, CHIR99021 GSK inhibitor/Wnt activator, DKK-1 Wnt inhibitor, EB embryoid body, FGF2 basic fibroblast growth factor, FLT3L FMS-like tyrosine kinase 3 ligand, HE hemogenic endothelium, IL-3 interleukin-3, IL-6 interleukin-6, SCF stem cell factor, TPO thrombopoietin, VEGFA Vascular Endothelial Growth Factor A