Next, CD43+ iHPCs were grown in serum-free differentiation medium (formulated in house) containing CSF-1, IL-34, and TGFβ1. By day 14, cells expressed the myeloid-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (Figure 1Aiii) demonstrating an early commitment toward microglial fate. Because this protocol yields large amounts of iMGLs, microglia development was studied in vitro as cells could be characterized every 4 days by flow cytometry. Day 14 early iMGLs were c-kit−/CD45+ (Figure 1C), suggesting commitment towards a myeloid lineage. Additionally, cells can be further subdivided into CD45+/CX3CR1− (A1) and CD45+/CX3CR1+ (A2) populations; similar to developing microglial progenitors identified in vivo (Kierdorf et al., 2013). CD45 expression was consistently monitored in developing iMGLs and compared to monocyte-derived macrophages (MD-Mφ). While CD45 expression increased with maturation, levels never reached that of macrophages (Figure 1D), consistent with murine development (Kierdorf et al., 2013). A small population of iMGLs (~10%) also expressed intermediate CD11b levels by day 14 that also increased as cells matured, but again never reached macrophage levels (Figure 1E, F).
