Extended Data Fig. 8: Changes in mtDNA sequence are enough to trigger cell competition.
a, Illustration of the process of derivation of the mESCs lines from mice that are hybrid between the wild-caught strains (BG, HB or ST) and the lab mouse (C57BL/6N). These hybrid mice were generated elsewhere16 by ooplasmic transfer: the zygote of a C57BL/6N mouse was injected with ooplasm from a wild-caught mouse (orange, HB pictured). Therefore, these hybrid mice contain the nuclear background of the C57BL/6N strain and the mtDNA of wild-caught strain and potentially C57BL/6N mtDNA (heteroplasmic mice strains). mESCs lines were derived from the hybrid mice and characterised. b-f, Characterisation of the derived cell lines by flow cytometry, during pluripotency, in comparison to the wild-type cell line used in previous experiments (E14, 129/Ola background). Heteroplasmy analysis of the derived mESC lines from the hybrid mice, indicating the percentage of wild-derived mtDNA (b). Cell granularity (internal complexity) given as median fluorescence intensity of SSc-A laser (c). Cell size given as median fluorescence intensity of FSc-A laser (d). Analysis of the expression of mitochondrial markers: representative western blot and quantification of markers of mitochondrial mass (ATPB, mt-CO1 and TOMM20) and mitochondrial dynamics (DRP1, MFN1and MFN2), relative to vinculin, in cells derived from hybrid mice (e). f, Representative histograms and quantification of median TMRM fluorescence, indicative of Δψm, for the hybrid cell lines derived, in comparison to the wild-type cell line used in previous experiments (E14, 129/Ola background). g-i, Cell competition assays between hybrid cell lines maintained in pluripotency culture conditions. The ratio of final/initial cell numbers in separate or co-culture is shown. j, Experimental design for RNA-Seq and gene set enrichment analysis (GSEA). The isolation of RNA from winner HB(24%) and loser BG(95%) cells was performed after three days in separate or co-culture conditions, once cells have been subjected to FACS to isolate the two populations form mixed cultures. Data shown as mean ± SEM of 3 independent experiments. See methods for details on statistical analysis.
