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

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GLIA_22343_sm_SuppFig1.tif3648KSupporting Information Figure 1. Morphological analysis of bone marrow derived CD117+Lin- and dendritic cells following their co-culture with neural cells. BM cells were generated from mice that express GFP under the human ubiqutin C promoter (U-GFP). The bone marrow cells were enriched based on surface CD117 (c-kit) and Lin- expression or differentiated to BMDCs as described in Materials and Methods. Images of U-GFP+CD117+Lin- enriched cells (A) co-cultured with primary glia for 1 day (Aa) or 7 days (Ab) and of U-GFP+BMDCs (B) co-cultured with OHSCs for 1 day (Ba) or 7 days (Bb). Representative cells from each of the images were then analyzed for morphological parameters using the Fiji software. GFP+ cells were traced (represented as black and white images) and their geometry measures were used to calculate their circularity. Number of branches and the total branch length were calculated using the skeletonize plugin. The table shown demonstrates that CD117+Lin- cells and to a further extent DCs acquired ramified morphologies at day 7 as compared with day 1 of their co-culture with neural cells, based on their reduced circularity and increased number and length of their branches.
GLIA_22343_sm_SuppFig2.tif6873KSupporting Information Figure 2. BMDCs up-regulate CX3CR1 when co-cultured with primary glia. BMDCs were generated from BM cells of GFP CX3CR1+/GFP mice and cultivated in the presence of GM-CSF for eight days. CX3CR1+/GFP BMDCs were cultured on primary glial cultures at a BMDC:glia ratio of 1:4 for 1, 3 or 7 days. (A) FACS analysis of CD11b (a), CD86 (b) and MHCII (c) expression by CD11c+ BMDCs in culture. (B) FACS analysis of GFP, indicating CX3CR1 expression by BM cells and BMDCs. (C) Live-cell imaging (a, b, c) and FACS analysis (d, e, f) of CX3CR1 expression in BMDC:primary glia co-cultures indicate that BMDCs up-regulate CX3CR1 and acquire microglia-like morphology. Scale bar represents 30 µm. Numbers in the graphs indicate the percentage of cells present within the M1 marker out of total gated cells and their MFI. The analysis shows one representative experiment out of 3 performed. (D-E) To demonstrate that GFP expression indeed reflects the expression of CX3CR1, microglia from adult CX3CR1+/GFP mouse brain and CX3CR1+/GFP BMDCs co-cultured with primary glia, were stained with FKN-Fc fusion peptide as described in Materials and Methods. (D) FACS analysis of CX3CR1+ cells derived from adult mouse brain without (a) and with (b) FKN-Fc+ staining. (E) FACS analysis of FKN-Fc+ staining of CX3CR1+/GFP BMDCs co-cultured with primary glia for 3 and 7 days.
GLIA_22343_sm_SuppFig3.tif1055KSupporting Information Figure 3. BMDC co-cultured with primary glia undergo limited proliferation and apoptosis. To determine whether BMDCs proliferate once co-cultured with primary glia, WT BMDCs were labeled with CFSE after eight days of culture, incubated in the absence or presence of mitomycin C for 2 h and then seeded on primary glia at a ratio of 1:4 for three days. As a positive control, WT BMDCs were stained with CFSE after three days of culture and continued to be cultured with GM-CSF for five more days. (A) FACS analysis of CFSE-labeled WT BMDCs. Arrows indicate populations with diluted levels of CFSE, representing proliferation cycles. FACS analysis of WT BMDCs co-cultured with primary glia without (b) or with (c) mitomycin C showing that BMDCs did not proliferate during the first three days of co-culture with primary glial cells. (B) To determine whether BMDCs undergo apoptosis while co-cultured with primary glia, GFP BMDCs were co-cultured with primary glia for 1, 3, and 7 days and stained for annexin V and PI, as described in Materials and Methods. Annexin V (a) and PI (b) FACS analysis of primary glial cells alone and their co-culture with GFP+BMDCs. Numbers in the graphs indicate the percentage of cells present in the quadrant out of total gated cells. The analysis shows one representative experiment out of 3 performed.
GLIA_22343_sm_SuppFig4.tif4976KSupporting Information Figure 4. BMDCs cultured with fibroblasts or peritoneal macrophages cultured with primary glia do not acquire microglial properties. CX3CR1+/-BMDCs were seeded on primary fibroblasts (A) or 3T3 cells (B) at a ratio of 1:4 for seven days. (A) Live-cell imaging of GFP-expressing cells (a) and a bright field image (b) of the co-culture. (B) Confocal microscopy analysis of fixed cultures for GFP (a, green) and CD11b immunolabeling (b, blue) in a co-culture of CX3CR1+/GFPBMDCs and 3T3 cells. (C) CX3CR1+/- or GFP macrophages were purified as described in Materials and Methods and then seeded on primary glial cultures at a ratio of 1:4 for seven days. FACS analysis of F4/80+ (a) and CD11b+ (b) CX3CR1+/- peritoneal macrophages. Live-cell imaging of GFP+ cells (c) and a bright field image of the co-culture (d). Live-cell imaging of U-GFP+ peritoneal macrophages co-cultured with primary glia on day 7 of the co-culture (e). The analysis shows one representative experiment out of 2 performed.
GLIA_22343_sm_SuppFig5.tif1096KSupporting Information Figure 5. CCR2 expression is down-regulated in BMDCs co-cultured with glia. CX3CR1+/GFP BMDCs were co-cultured with primary glia at a BMDC:glia ratio of 1:4 for 7 days. (A) Representative FACS dot plot images of CCR2 expression in CX3CR1+/GFP BMDCs (a), primary glia (b) and in DMSO- or SB-treated co-cultures (c and d, respectively). (B) Quantification analysis of the percentage of CCR2+GFP- cells (R03 gate, black bars) and GFP+ cells (R04 gate, gray bars) out of total gated cells in primary glia and in DMSO- or SB-treated co-cultures. Statistical analysis was performed with GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA). To test the hypothesis that cell fractions varied with treatment (DMSO or SB), a general linear 2-way ANOVA model was used. The Bonferroni's correction was used to assess the significance of the predefined comparisons. According to these data, treatment with SB resulted in a significantly reduced numbers of GFP+ cells concomitant with increased numbers of CCR2+GFP- cells. Whereas the CCR2 MFI of CX3CR1- cells (RO3) was 69+2.45 and 74.7+4.25 in DMSO- and SB-treated co-cultures, respectively, CCR2 MFI of the GFP+ cells (RO4) was reduced to 17.39±0.72 and 24.2±1.6, respectively. Overall, these results demonstrate that CCR2 is down-regulated in BMCDs differentiating to CX3CR1+ microglia-like cells.

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