Functional Ramifications for the Loss of P-Selectin Expression on Hematopoietic and Leukemic Stem Cells
Figure 2
Selp−/− donor cells exhibit diminished homing velocities but longer-term repopulation advantages over controls.
(A,B) CD45.2 LSK cells derived from Selp−/− and UBC-GFP mice were sorted by sterile technique and mixed in a 1∶1 ratio. (A) Peripheral blood was extracted from recipients at 30 d (N = 6), 45 d (N = 6), 86 d (N = 5), and 120 d (N = 3) post-transplantation and subjected to FACS analysis (top). The CD45.2 cell population was assayed, and the percentage of GFP+ (UBC-GFP) and GFP- (Selp−/−) cells were determined. Data are represented as the mean ± SEM. One sample t-tests (theoretical means = 50%, assuming no competitive advantage) were applied to the data. Statistically significant differences were not observed at 30 d (P = 0.9639) but became apparent at 45 d, 86 d, and 120 d (P = 0.0113). (B) The percentage of HSC-containing LSK cells, and specifically CD34− LSK LT-HSCs, either GFP+ or GFP−, in bone marrows from recipients at 120 d (N = 3) post-transplantation were determined by FACS analysis. Data are represented as the mean ± SD. HSC-containing LSK cells and CD34− LSK LT-HSC derived from Selp−/− exhibited a competitive advantage. (C) The HSCs of Selp−/− self-renew significantly more than WT HSCs in 2nd transplantation experiment. The bone marrow cells from primary recipient were collected and transplanted into secondary recipients at 30 d post primary transplantation. The percentage of LSK cells and CD34− LSK LT-HSC cells in the bone marrow of the recipients (Selp−/−) cells were determined at 120 d post 2nd transplantation. Data are represented as the mean ± SD (N = 4). (D) Homing assay reveals decreased homing velocity for Selp−/− cells relative to UBC-GFP controls. Bone marrow cells from Selp−/− or WT mice (both CD45.2 background) were mixed in a 1∶1 ratio with UBC-GFP cells (CD45.2) and injected into CD45.1 recipients. Three hours post-injection, recipients were sacrificed and the percentages of CD45.2/GFP+ and CD45.2/GFP- cells were measured by FACS (representative FACS, left panel). The average ratios of WT to UBC-GFP or Selp−/− to UBC-GFP cells are presented. Selp−/− cells exhibited a significant homing defect relative to controls. (E) HSC homing assay reveals a decreased homing velocity for Selp−/− LSK cells relative to WT controls. Bone marrow cells from Selp−/− or WT mice were respectively injected into CD45.1 recipients (HSCs were normalized by FACS before injection). Three hours post-injection, recipients were sacrificed and the percentages of CD45.2+ LSK were measured by FACS (representative FACS, left panel). The average cell number (LSK) of WT or Selp−/− are presented. (F) Cell cycle analysis of WT and Selp−/− HSCs. Bone marrow cells were isolated from Selp−/− or WT mice. LSK cells were stained with Hoechst blue, and LSK cells in S+G2M phase of the cell cycle were examined by FACS. No significant differences were observed. For each experiment, the following denote the statistical significance observed: 0.01<P<0.05 = *; 0.001<P<0.01 = **; P<0.001 = ***.