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Supplemental material, MSO894615 Supplemental Material for Is multiple sclerosis progression associated with the HLA-DR15 haplotype? by Klarissa Hanja Stürner Inessa Siembab Gerhard Schön Jan-Patrick Stellmann Nika Heidari Boris Fehse Christoph Heesen Thomas H Eiermann Roland Martin Binder Thomas MC in Multiple Sclerosis Journal—Experimental, Translational and Clinical

Neurogenesis is altered in neurodegenerative disorders, partly regulated by inflammatory factors. We have investigated whether microglia, the innate immune brain cells, regulate hippocampal neurogenesis in neurodegeneration. Using the ME7 model of prion disease we applied gain- or loss-of CSF1R function, as means to stimulate or inhibit microglial proliferation, respectively, to dissect the contribution of these cells to neurogenesis. We found that increased hippocampal neurogenesis correlates with the expansion of the microglia population. The selective inhibition of microglial proliferation caused a reduction in neurogenesis and a restoration of normal neuronal differentiation, supporting a pro-neurogenic role for microglia. Using a gene screening strategy, we identified TGF? as a molecule controlling the microglial pro-neurogenic response in chronic neurodegeneration, supported by loss-of-function mechanistic experiments.By the selective targeting of microglial proliferation we have been able to uncover a pro-neurogenic role for microglia in chronic neurodegeneration, suggesting promising therapeutic targets to normalise the neurogenic niche during neurodegeneration.

Figure S1. Karyotyping. One clone from BJ cell line and two clones from C25-old cells sorted by flow cytometry, cultured for a few passages and karyotype analysed. Karyotyping indicated no chromosomal anomalies in the three sub-clones. Figure S2. Flow cytometry and fluorescence microscopy of RGB-marked cell line 293 T at different time points. Full data for cell line 293 T from Fig. 3. Left to right in each panel: green plotted against blue channel, green against red and blue against red. MOI 4 used to transduce 293 T cells. (A) Clonal dynamics in T75-flask. (B–D) Clonal dynamics in one individual well of a six-well cell culture plate over 25 weeks. (E) Stacked area plots displaying time course of proportion of each possible combination of colours (no colour, red only, green only, blue only, red and green, red and blue, blue and green, all three colours) to sum of all cells. Note similar dent in all plots at week 23 (right panel), probably due to technical reasons. Left panel shows plots with interpolated count at week 23. (F) Fluorescence microscopic images of four cultures in (A)–(D) at week 25. Images taken 3 days after replating. Figure S3. Flow cytometry and fluorescence microscopy of RGB-marked cell line K562 at different time points. Full data for cell line K562 from Fig. 3. Left to right in each panel: green plotted against blue channel, green against red and blue against red. MOI 16 used to transduce K562 cells. (A) Clonal dynamics in T75-flask. (B–D) Clonal dynamics in one individual well of a six-well cell culture plate over 25 weeks. (E) Stacked area plots displaying time course of proportion of each possible combination of colours (no colour, red only, green only, blue only, red and green, red and blue, blue and green, all three colours) to sum of all cells. Note similar dent in all plots at week 23 (right panel), probably due to technical reasons. Left panel shows plot with interpolated count at week 23. (F) Fluorescence microscopic images of the four cultures shown in (A)–(D) at week 25. Images taken 3 days after replating. Figure S4. Flow cytometry and fluorescence microscopic images of RGB-marked primary human dermal fibroblast cultures at different time points. Full data set for primary fibroblasts from Fig. 4. Left to right in each panel: green plotted against blue channel, green against red and blue against red. MOI 1, 2, 3 and 10 used for transduction and four cultures kept separately afterwards. (A) Clonal dynamics analysed by flow cytometry only (p2). (B) Clonal dynamics analysed by flow cytometry and imaged by fluorescence microscopy (further passages). Insets display higher magnification (PDF 24080 kb)

Background Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. Methods We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. Results While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. Conclusion Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0668-x) contains supplementary material, which is available to authorized users.

eGFP expression and clonal kinetics in the BcrAbl_bulk culture. Figure S2: Overlap of barcodes from plasmid library and first sample. Figure S3: Clonal kinetics in the eGFP-positive cultures after sorting. Figure S4: Spleens from diseased (and control) animals. Figure S5: Read counts for Klf6 and Sprouty1. Figure S6: Comparison of samples. Figure S7: KEGG enrichment analysis. Table S1: Spleen weights and eGFP expression in the diseased animals. Supplementary Model Description. Table S2: Model Parameters. Table S3: Oligonucleotides. (PDF 7697Â kb)