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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 27 NOV 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 12, pages 2657–2671, December 2012
How to Cite
Cusulin, C., Monni, E., Ahlenius, H., Wood, J., Brune, J. C., Lindvall, O. and Kokaia, Z. (2012), Embryonic Stem Cell-Derived Neural Stem Cells Fuse with Microglia and Mature Neurons. STEM CELLS, 30: 2657–2671. doi: 10.1002/stem.1227
Author contributions: C.C. and E.M.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing, H.A., J.W., and J.C.B.: collection and assembly of data and data analysis and interpretation; O.L. and Z.K.: conception and design, financial support, data analysis and interpretation, and manuscript writing. C.C. and E.M. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS November 7, 2012.
- Issue published online: 27 NOV 2012
- Article first published online: 27 NOV 2012
- Accepted manuscript online: 7 SEP 2012 01:05PM EST
- Manuscript Accepted: 10 AUG 2012
- Manuscript Received: 22 FEB 2012
- Swedish Research Council, European Union projects StemStroke. Grant Number: (037526)
- TargetBraIn. Grant Number: (279017)
- AFA Foundation, and Swedish Government Initiative for Strategic Research Areas (StemTherapy)
Additional Supporting Information may be found in the online version of this article.
|sc-12-0183_sm_SupplFigure1.pdf||187K||Figure 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.tif||2572K||Figure 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.pdf||166K||Figure 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.pdf||70K||Figure 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.pdf||212K||Figure 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.pdf||129K||Figure 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.pdf||69K||Figure 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.pdf||145K||Figure 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.pdf||55K||Supplemetary Table 1|
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