Figure 1.

Quiescent Hematopoietic Stem and Progenitor Cells Express High Levels of SCA-1

(A–C) R26^rtTA/Col1A1^H2B-RFP mice were DOX-pulsed, chased for either 4, 6, 13, or 19 weeks, and HSPC populations were analyzed by flow cytometry for retention of H2B-RFP (see also Figures S1A–S1D). (A) Representative histograms of BM HSCs (LSK CD48^−/loCD150⁺, upper row) or MPPs (LSK CD48^−/loCD150⁻, middle row) from mice (n = 9) chased for 19 weeks are shown. H2B-RFP⁺ label retaining cells (black lines) were overlaid on to the total parental population (solid gray histograms). Antigen median fluorescence intensities (MFI, lower row, normalized to the mean MFI of HSCs) of the total or H2B-RFP⁺ populations were determined and compared. (B) Representative histograms for SCA-1 expression of BM HSCs (left column) and MPPs (middle column) isolated from R26^rtTA/Col1A1^H2B-RFP mice chased for either 4 (upper row, n = 2), 6 (middle row, n = 4), or 13 weeks (lower row, n = 8) are shown and H2B-RFP⁺ (black lines) and H2B-RFP⁻ cells (solid gray histograms) were overlaid. SCA-1 MFIs (right column, normalized to total HSC population) of H2B-RFP⁻ and H2B-RFP⁺ HSPCs were compared. (C) Representative SCA-1 expression histograms of BM HPC-1 (LSK CD48^hiCD150⁻, left column) or HPC-2 cells (LSK CD48^hiCD150⁺, middle column) from R26^rtTA/Col1A1^H2B-RFP mice (as in B) are shown. H2B-RFP⁺ populations (black lines) were overlaid onto the respective parental population (solid gray histograms). SCA-1 MFI (right column, mean MFI of total HPC-1 set to 1) of total and H2B-RFP⁺ populations was calculated.

(D) BM of Ki67-RFP^ki/wt mice (n = 4, see also Figures S1E–S1G) was analyzed by flow cytometry. Representative contour plots of HPCs-1, MPPs, and HSCs are shown (upper row; dotted line shows threshold for KI67-RFP gating, set according to Ki67-RFP^wt/wt control). Lower row compares SCA-1 expression between KI67-RFP⁺ cells (solid gray histograms) and KI67-RFP⁻ (black lines) cells among the respective parental population. Right data plot quantifies SCA-1 MFI of HSPC populations separated by KI67-RFP expression. SCA-1 MFI was normalized to the mean MFI of the total HSC population, which was set to 1.

Means ± SD are shown throughout the figure. Significance was calculated by paired Student's t test and Bonferroni-Holm error correction for multiple testing. ^∗p = 0.01–0.05, ^∗∗p = 0.001–0.01, ^∗∗∗p < 0.001; ns, not significant. See also Figure S1.