Fig. 1

Differentiation of hiPSCs into microglia-like cells (iMG). A. Schematic overview of two-step differentiation of hiPSCs into iMG. In the first step the hiPSCs are differentiated into hematopoietic progenitors (HPCs) using media A (days 0–3), followed by media B (days 3–12) from a commercial kit. At day 12, cells exhibit CD34⁺, CD43⁺, and CD45⁺ expression and are transferred to poly-L-lysine coated plates containing media C⁺⁺⁺ supplemented with astrocyte-conditioned media. (HPCs can be cryopreserved at this level). By day 16, PU.1 transcription factor emerges, and non-adherent round IBA1/TMEM119⁺ iMG appear on day 20. IBA1/TMEM119⁺ iMG can be sequentially harvested up to six times and upon further 2–5 days of culturing in defined media, they acquire homeostatic properties. The upper panel shows the representative images of differentiating iPSCs, HPCs, floating iMG (black arrow), and iMG cultured in near homeostatic conditions. B. qPCR data shows the expression of microglial signature genes in iMG derived from three different iPSCs lines. Color and style denotes p-values and iPSC line, respectively, with the X-axis displaying the log2 fold change. C-D. Flow cytometry analysis shows the expression of CD11B and CD45 on day 20 iMG. Red histograms represent the isotype control staining, blue histograms show the CD11B and CD45 staining. E. Immunocytochemistry (ICC) of TEMEM119 expression under near homeostatic conditions in iMG derived from indicated iPSC lines. Scale bars represent 10 μm. F. Displays relative percentage of TMEM119 positive and TMEM119 negative cells in iMG cultures differentiated from three independent iPSC lines