Fig. 1

Differentiation of hiMGLs from hiPSCs through overexpression of PU.1 transcription factor. A Schematic diagram of the tet-on inducible expression vector of SPI1 and plasmids expressing hyperactive piggyBac transposase (HyPBase) or reverse tetracycline transactivator (rtTA). B Protocol for generating hiMGLs from hiPSCs. For PU protocol, doxycycline (DOX) was added into the medium from days 6 to 18. C qRT-PCR data about the expression level 1 day after the DOX was added (day 7). Compared with the groups with no DOX (Ctrl), SPI1, as well as congruent βGeo, was strikingly upregulated after DOX treatment for 24 h (n = 3 independent clones). There was no observed difference of the 3′UTR of the SPI1 gene, which is not included in the SPI1 expression plasmid, indicating that the high expression level of SPI1 on day 7 was exogenous. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. All data are expressed as mean ± SEM. SEM, standard error of the mean. D Bubble-like structures were observed in the PU protocol, not in the CK protocol. Scale bar, 200 μm (left) and 50 μm (right). E More than three times of hiHPC (microglia progenitor cells) could be harvested (days 18, 22, 26) by using the PU protocol, compared to the CK protocol (n = 3 independent experiments). F Representative images of hiMGLs. iMGLs acquired more ramified morphology 3 weeks after re-plating of hiHPCs (day 39) than those from the shorter culture (day 25). Scale bar, 50 μm (upper) and 25 μm (lower)