Fig. 1

Schematic representation for the generation of iMphs from PSCs using different protocols. In all the protocols, the differentiation of PSCs into iMphs goes through four main stages as described. Embryoid Body-Spontaneous (EB-S) Step-1, PSCs culturing- PSCs are cultured and expanded on MEFs. Stage 1, M/HE stage- The mesoderm or hemogenic endothelium (M/HE) is induced through the EB formation in ultra-low adhesive (ULA) culture dishes. Stage 2 and Stage 3, HP and MY stages, the EBs formed in stage 1 are transferred to matrix-coated plates and cultured in medium supplemented with IL-3 and M-CSF under normoxia conditions. The floating monocyte-like cells (iMCs) are collected and transferred to new culture plates for their terminal differentiation. Stage 4, MF terminal differentiation- The cells collected from stage 3 are cultured using RPMI medium supplemented with M-CSF for their terminal differentiation into macrophages-derived from iPSCs (iMphs). Embryoid Body-Factors (EB-F) HP + MY: Step-1, PSCs culturing- PSCs are cultured and expanded on matrix-coated culture plates. Stage 1, M/HE stage- The mesoderm/HE specification in EBs is directed by externally supplied factors under normoxia condition. Stage 2 and Stage 3, HP and MY stages, the EBs from stage 1 are transferred to new plates and cultured in medium supplemented with IL-3 and M-CSF. Similar to EB-S protocol, the floating cells are collected and transferred to new culture plates for their terminal differentiation. Stage 4, MF terminal differentiation- The cells collected from stage 3 are cultured using RPMI medium supplemented with M-CSF for their terminal differentiation into iMphs. Embryoid Body-Factors (EB-F) HP → MY Step-1, PSCs culturing- PSCs are depleted from the MEFs before subjecting to differentiation. Stage 1, M/HE stage- The mesoderm/HE specification in EBs is directed by combination of exogenous factors under normoxia or hypoxia conditions. Stage 2, HP stage- The EBs obtained from stage 1 are transferred to either ULA or matrix-coated plates and cultured in the presence of specific exogenous factors to induce HP stage. Stage 3, MY stage- In the MY stage, the composition of exogenous factors is modified for the generation of iMCs. Stage 4, Stage 4, MF terminal differentiation- The floating iMCs cells are collected from stage 3 and cultured with M-CSF for their terminal differentiation into iMphs. Embryoid Body-independent 2D-Factors Step-1, PSCs culturing- PSCs are cultured on Matrigel-coated plates. Stage 1, M/HE stage- The M/HE is induced by culturing cells in M/HE-specific factors on matrigel coated plates under normoxia or hypoxia condition. Stage 2, HP stage- The HP specification is achieved in the presence of HP-specific factors. Stage 3, MY stage- For MY differentiation, the cells are either transferred to ULA plates or matrigel-coated plates and cultured under a set of exogenous factors for further differentiation into iMCs. Stage 4, MF terminal differentiation-The floating iMCs cells are collected from stage 3 and cultured with M-CSF for their terminal differentiation into iMphs