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CD27, CD201, FLT3, CD48, and CD150 identify HSC in mice
CD27+ CD201+ FLT3- cells that were not in the CD48- CD150+ gate (NOT GATE) (Figure 5H, isotype controls in Online Supplementary Figure S5). We transplanted serial dilutions of these two populations (Figure 5H) into sub- lethally irradiated (2.5 Gy) female NSG recipient mice together with 100,000 lethally irradiated whole BM as car- rier cells. At 8, 12, and 16 weeks a small amount of blood was lysed for longitudinal analysis of donor engraftment by genomic quantitative polymerase chain reaction using primers specific for the Y chromosome Sry gene compared to biallelic mouse Il6 gene. In preliminary experiments, we validated this method of quantifying relative male cell
number by mixing a known amount of male vs. female cells to demonstrate that the assay readout reflected the linear dilution series (Online Supplementary Figure S6). A level of >1% donor male cells at the 18-week harvest point was considered to be a successful reconstitution of the host (Online Supplementary Table S4).
In transplanted recipients, we measured chimerism between 8 and 18 weeks. There was robust long-term male donor chimerism in recipients that received 50 or 150 CD48- CD150+ gated cells whereas there was a very “low” frequency of long-term chimerism in recipients of NOT GATE cells (Figure 5I, J). Poisson distribution analysis
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Figure 2. Complementarity of CD27 and CD201 with FLT3, CD48 and CD150 staining in different mouse strains. The gating strategy is shown in Online Supplementary Figure S1. (A-C) Lin- KIT+ CD27+ CD201+ FLT3- bone marrow (BM) cells were gated and analyzed for CD150 and CD48 expression in C57BL/6 (A), NOD-scid (B) and NSG (C) mice. (D, E) Frequency (D) and total number (E) of Lin- KIT+ CD27+ CD201+ FLT3- CD48- CD150+ cells per femur in each strain. (F) Overlay of CD48 expression in Lin- KIT+ CD27+ CD201+ FLT3- BM cells from C57BL/6 (grey shaded), NOD-scid (blue) and NSG (red) mice. (G) Geometric mean fluorescence intensity of CD48 on Lin- KIT+ CD27+ CD201+ FLT3- cells in the three mouse strains. Data are the mean ± standard deviation of five mice per group. P values were calculated by analysis of variance with Tukey corrections for multiple comparisons, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
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