Additional Supporting Information may be found in the online version of this article.

sc-12-0183_sm_SupplFigure1.pdf187KFigure S1. Fused NS/microglia cells maintain proliferative activity. (A−C) Live cell image of proliferating GFP+/RFP+ fused NS/microglia cells. Virtually all cells express GFP+ and RFP+. (D−G) After co−culture of microglia and NS cells and subsequent sorting, fused GFP+/RFP+ cells continue to proliferate, as demonstrated by staining for phosphohistone−3 (p−H3). Similarly, fused cells in co−culture of rat primary microglia and mouse ES cell−derived NS cells express Ki67 (H−K). Some double−positive fused cells (L−S) exhibit two distinct nuclei as visualized by nuclear stain (N and R). Scale bars: AC= 50 μm; D−O=15 μm; P−S =10μm.
sc-12-0183_sm_SupplFigure2.tif2572KFigure S2. Sorted RFP−only, presumed microglia cells do not differentiate to neurons. After co−culturing RFP+ microglia and GFP+ NS for 3 days and sorting cells that were RFP+ only, prospectively all microglia cells, do not express MAP2 when exposed to neuronal differentiation protocol. Scale bar=20μm
sc-12-0183_sm_SupplFigure3.pdf166KFigure S3. NS cells and fused cells exhibit similar electrophysiological characteristics. (A) Representative trace of an ‘overshoot’ action potential generated by NS cells (held at ∼ −75 mV) in response to a 180 pA depolarizing current step (top). Below, representative trace of an inward current induced by a voltage−step from −90 mV to −10 mV. (B) Representative traces of an ‘overshoot’ action potential and inward current observed in fused cells using the protocol described. (C−F) A fused recorded cell filled with biocytin and subsequently stained for GFP, RFP and Hoechst. Scale bar=20μm.
sc-12-0183_sm_SupplFigure4.pdf70KFigure S4. Fused NS/microglia cells display functional properties of microglia. (A) Quantitative PCR shows that activation in culture of fused cells by LPS leads to almost 2−fold increased expression of iNOS and TNF and that IL−4/IL−13 induce 8− and 53−fold increase of CD206 and arginase, respectively. (B) Similar responses are detected in non−fused microglia but levels of expression are much higher.
sc-12-0183_sm_SupplFigure5.pdf212KFigure S5. Microglia and NS cells exhibit membrane molecules required for PS−dependent fusion. (A−C) Proliferating living NS cells expose PS without any sign of apoptosis. AnnexinV binds only to PS located in the outer leaflet of the membrane. (E−F) Microglia express CD36, the scavenger receptor which is involved in macrophage fusion.
sc-12-0183_sm_SupplFigure6.pdf129KFigure S6. Fused NS/microglia cells fuse with mouse cortical cells. (A) Bivariate pseudocolor plot of a co−culture of CellVue+ mouse primary cortical culture and fused GFP+ NS cells and RFP+ microglia cells. The gate represents the population of triple−positive cells (CellVue+ GFP+ and RFP+) which has been sorted. (B) FACS plot of fused GFP+/RFP+/CellVue+ cells. (C) Purity reanalysis of sorted triple−positive cells. At least 100 cells were recorded, showing that 91.1% of all cells were GFP+/RFP+ and 97.1% of them were also positive for CellVue (D). (E−H) Triple−positive cells after replating and culture in neuronal differentiation medium. At 7 days, cells express the neuronal marker MAP2.
sc-12-0183_sm_SupplFigure7.pdf69KFigure S7. Microglia and fused NS/microglia cells do not fuse with cells of human origin. (A−H) Human fetal cortex−derived NS cells (B) or human iPS cell−derived lt−NES cells (F) do not fuse with co−cultured microglia (A and E). (I−L) Confocal images of human primary cortical cells, immunopositive for the human cytoplasmic marker SC121 (I), co−cultured with GFP+/RFP+ fused NS/microglia cells (I and J). Fusion between fused cells and primary cortical cells has not been observed. Scale bars: D, H=20 μm, L=20μm.
sc-12-0183_sm_SupplFigure8.pdf145KFigure S8. Three−dimensional reconstruction reveals presence of two nuclei inside soma of some fused cells. (A−D) Pyramidal neuron indicated by arrow bears two nuclei, as demonstrated by the 3D reconstruction in (E) Every column represents a different plane used for bi−dimensional representation of the cell. Bottom row indicates the angle (plane) of observation of confocal images in corresponding column. The two nuclei are circled in white. Scale bar D=50μm, E=10μm.
sc-12-0183_sm_SupplTable1.pdf55KSupplemetary Table 1

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