Fig. 1.

Mutant HTT does not affect the differentiation of human PSC-derived microglia. a PSC lines expressing HTT of different polyglutamine repeat lengths were differentiated to a microglial fate. They were assessed for the expression of genes associated with microglial identity, both to confirm generally the robustness of the methods used across multiple lines and to test specifically for any mutant HTT-dependent effects on differentiation. The macrophage markers, TREM2 and IBA1, were significantly elevated in each of the lines tested compared with iPSCs and, for the former at least, primary human monocytes. TMEM119, a gene whose expression is associated with microglia/tissue-resident macrophages when compared with their systemic, monocyte-derived counterparts, was elevated in each of the lines tested compared with iPSCs, primary monocytes and monocyte-derived macrophages. P2RY12, a gene whose expression also is associated with microglia/tissue-resident macrophages, was far more variable in its expression yet was significantly elevated in some of the lines. Taken together, these data indicate the robustness of the methods used across all the lines, but do not show any mutant HTT-dependent effects on differentiation. b IsoHD PSC lines expressing HTT of different polyglutamine repeat lengths were differentiated multiple times to a microglial fate. Quantification of the numbers of cells expressing protein markers of microglial identity was undertaken by high-content immunofluorescence microscopy. This showed that the cultures were almost entirely enriched for microglial-like cells expressing TREM2, IBA1, TMEM119 and PU1. There were no HTT polyglutamine repeat-dependent differences in proportions of cells that were positive for these protein markers. Data are presented as mean ± SEM, analysed by one-way ANOVA with Tukey’s multi-comparison post-test (*/#/^ p < 0.05, **/##/^^ p < 0.01, ***/###/^^^ p < 0.001; where * = vs iPSCs, # = vs primary monocytes, ^ = vs monocyte-derived macrophages)