Fig. 4. c-kit^Cre mice fail to label resident CSCs.

a Photographs of representative adult c-kit^MCM and c-kit^CreGFPnls/+ mice showing the typical W-mutation-determined piebald and white belly spot as compared with c-kit^+/+ C57BL/6J mouse. b The testis from 2-month-old kit^MCM and c-kit^CreGFPnls/+ show a significant size reduction compared to c-kit^+/+ mice. c Cell growth curve of freshly isolated (P1) W^CreCSCs vs. wtCSCs over 96 h. n = 7 biological replicates, *p < 0.05 vs. wtCSCs (Kruskal–Wallis test). d Colony number of 100 cloned wtCSCs vs. W^CreCSCs in Methocult. n = 7 biological replicates, *p < 0.05 vs. wtCSCs (Student’s t-test). On the right, representative light microscopy images of a colony from wtCSCs and W^CreCSCs, respectively. n = 6 plots biological replicates. Scale bars = 1000 µm. e GFP expression (from the c-kit^CreGFPnls allele) in the monocyte–lymphocyte gate of total bone marrow (BM) cells population and in total c-kit^pos BM cells from 2-month-old c-kit^CreGFPnls/+ mice. Less than 25% of the total c-kit^pos BM cells express GFP in c-kit^CreGFPnls/+ mice. Concurrently, <35% of the lineage-negative (Lin^neg) c-kit^pos BM cells (including HSCs) express GFP in c-kit^CreGFPnls/+ mice. f GFP expression (from the c-kit^CreGFPnls allele) in cardiomyocyte-depleted cardiac cells (left) and in cardiac c-kit^pos cardiac cells (right) from 2-month-old c-kit^CreGFPnls/+ mice. Less than 10% of these cells express GFP. Right, confocal microscopy representative images of cardiac cross-sections showing that GFP nuclear expression is expressed only in some (20 ± 3%) of the c-kit-expressing cardiac cells. Scale bar = 20 µm. (e, f representative of n = 5 BM/Hearts). g Mice KI-ed within the first exon of the c-kit locus to express Cre recombinase and GFP with a nuclear localization sequence (eGFPnls) behind an internal ribosome entry site (IRES) (c-kit^CreGFPnls or c-kit^Cre) were crossed with B6.129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (abbreviated as R26^{floxed-dTomato}) Cre-reporter mice, which harbour a targeted mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato), which is expressed following Cre-mediated recombination. Approximately 20% of total cardiac cells and ~60% of c-kit^pos cardiac cells from these mice are dTomato^pos. Approximately 70% of lineage-committed endothelial/mast cell (CD45^pos/CD31^posc-kit^pos) cardiac cells are recombined to express dTomato, whereas <10% of the CSC-enriched CD45^negCD31^neg/c-kit^pos were recombined to become dTomato^pos (g representative of n = 5 hearts). All data are mean ± SD