Engraftment and Characterization of Human iPSC and ESC-Derived Microglia in Mouse Brain

PMP were collected from supernatant and suspended as single cells at a final concentration of 100,000 cells per μl in PBS. The cells were then injected into the brains of P0 Rag2^−/−hCSF1 immunodeficient mice (C;129S4-Rag2^tm1.1FlvCsf1tm1^(CSF1)FlvIl2rg^tm1.1Flv/J, The Jackson Laboratory). The precise transplantation sites were bilateral from the midline = ±1.0 mm, posterior bregma = −2.0 mm, and dorsoventral depth = −1.5 and −1.2 mm (Fig. 1c). The mouse pups were placed on ice for 5 min to anesthetize. The pups were injected with 0.5 μl of cells into each site (total four sites), using a digital stereotaxic device (David KOPF Instruments) that was equipped with a neonatal mouse adapter (Stoelting)^{16 (link)}. The pups were weaned at 3 weeks and were kept up to 6 months. All animal work was performed without gender bias with approval of the Rutgers University Institutional Animal Care and Use Committee.
Both hiPSC- and hESC-derived PMPs were transplanted into mouse brains. Both engrafted hiPSC- and hESC-derived microglia were analyzed, including characterization of their marker expression (Fig. 1), morphological changes along brain development (Fig. 2), and their phagocytic functions under homeostatic condition (Fig. 3), as well as toxin-induced demyelination condition (Fig. 6). For the quantification, we pooled the data collected from both hiPSC- and hESC-derived microglia. In the single-cell RNA-sequencing experiment, we only used the animals received transplantation of hiPSC-derived microglia. For cuprizone-induced demyelination, 3-month-old chimeric mice were fed with cuprizone diet (Sigma-Aldrich, 0.2%) or control diet for 4 weeks.