Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.

已经发布了多种协议，用于从hESC / iPSC生成iMGL。迄今为止，还没有指南可以帮助研究人员确定最适合小胶质细胞研究的方法。为了建立便于将来进行小胶质细胞研究的框架，我们首先在使用三个已公开的数据集衍生的iMGL之间进行了比较转录分析，这使我们能够建立最能代表善意人类小胶质细胞的基线方案。其次，使用CRISPR标记具有纳米荧光素酶和tdTomato的经典小胶质细胞标记物CX3CR1，我们生成并在功能上验证了报告基因ESC系。最后，使用该细胞系，我们证明了iMGL前体与人神经胶质细胞和神经元的共培养可增强iMGL的转录相似性，从而离体小胶质细胞。总之，我们全面的分子分析和报告细胞系对于寻求使用iMGL进行疾病建模和药物筛选研究的神经生物学家来说是有用的资源。