Study of microglia has been largely restricted to non

Study of microglia has been largely restricted to non-human models (mostly mouse), since availability of fresh primary human microglia is very limited and they cannot be propagated. Moreover, microglia rapidly lose their unique identity when removed from the inos inhibitor environment and cultured in monoculture in vitro (Butovsky et al., 2014). Transformed microglial-like cell lines are by definition highly proliferative and therefore not a good model for understanding a predominantly non-proliferating, differentiated cell type. There is therefore a need for practical, authentic human microglial cellular models. However, only recently has the ontogeny of microglia been established to inform appropriate modeling. In mice, two waves of embryonic macrophages are produced in the yolk sac blood islands at embryonic day 7.5 (E7.5) and E8.25, and the first wave migrate into the developing brain and differentiate to microglia (Ginhoux et al., 2010; Gomez Perdiguero et al., 2015; Hoeffel et al., 2015; Palis et al., 1999). These yolk sac-derived macrophages are Myb independent but dependent on PU.1 and Irf8 (Kierdorf et al., 2013; Schulz et al., 2012). Hematopoietic stem cells (HSCs), in contrast, derive from the aorto-gonado-mesonephros region at day E10.5, populate the fetal liver and bone marrow, and give rise to adult blood cells from HSCs in bone marrow niches, which are dependent on Myb for their renewal. Myb independence, therefore, distinguishes yolk sac-derived macrophages from adult, definitive, blood monocyte-derived macrophages. Microglia in the developing brain proliferate locally at a low rate and are not normally replaced by other monocytes and macrophages from outside the brain, in contrast to most other tissue-resident macrophages (which also initially originate from yolk sac-derived macrophages, but are partially or fully replaced by fetal liver- or blood monocyte-derived macrophages [Bain et al., 2014; Calderon et al., 2015; Epelman et al., 2014; Guilliams et al., 2014; Hoeffel and Ginhoux, 2015; Tamoutounour et al., 2013]). In the brain, interleukin-34 (IL-34) is an alternative CSF1R ligand supporting microglia survival and differentiation (Greter et al., 2012), and microglia adopt an increasingly ramified morphology and continued maturation far beyond birth. In humans there are few opportunities to investigate the ontogeny of microglia, but it is assumed that the processes are analogous to those in mice. Yolk sac-derived macrophages appear at E17 (Tavian and Peault, 2005), enter the brain from E31 onward (Rezaie et al., 2005; Monier et al., 2007), and mature together with neurons to fully functional ramified microglia (Figure 1A). Human cortical neurons show spontaneous electrical activity after microglia invasion, from gestation week 20 onwards (Moore et al., 2011). We aimed to recapitulate the in vivo developmental pathway of microglia in vitro, using human induced pluripotent stem cells (iPSCs). These have the advantages of limitless self-renewal and normal karyotype, and can be directed to terminally differentiated cell types. They can be derived from patients (retaining the patient\'s genetic background) and are amenable to gene editing, enabling sophisticated interrogation of genes of interest. To recapitulate the development of yolk sac-derived macrophages, we use our previously established, straightforward, highly efficient, serum- and feeder-free protocol for deriving PSC macrophages (Karlsson et al., 2008; van Wilgenburg et al., 2013). We have recently directly demonstrated that these derive from MYB-independent, RUNX1- and PU.1-dependent precursors, characteristic of yolk sac-derived macrophages (Buchrieser et al., 2017; Vanhee et al., 2015). Here, we co-culture them with iPSC cortical neurons (Shi et al., 2012), in medium optimized for survival and functionality of both neurons and microglia. The resulting co-cultures are stable for many weeks, express relevant microglia markers (including key disease-related genes), upregulate pathways relating to homeostatic functions, and downregulate pathogen-response pathways. They are phagocytic, display highly dynamic ramifications, respond to activation by clustering and adoption of ameboid morphology, and produce cytokine profiles that are specific to co-culture versus monoculture.