Macro-anatomical mapping of reconstituting stem cell niches. (A) Representative FACS plots showing the gating strategy used to analyze donor-derived chimerism 9 days (identical strategy used at day 21) posttransplantation (left). Gating strategy used to analyze HSPC homing efficiency at 16 hours (right panel). In order to ensure accuracy of the analysis at each time point, the precise positioning of the different gates was first performed on the recipient (CD45.2) HSPC subpopulations and then pasted on the donor cells (CD45.1). (B-C) Donor-derived chimerism 9 and 21 days postintravenous infusion of 2.3 × 105 to 10 × 105 lineage-depleted HSPCs (n = 10 and n = 8, respectively, 2 independent experiments). Total chimerism level (B) and frequency of donor-derived HSPCs as a frequency of total CD45+ BM cells (C). (D) Homing efficiency: 16 hours postintravenous infusion of 17 × 106 to 36 × 106 congenic CD45.1 BM-MNCs, diaphyses, epiphyses, and calvaria of the CD45.2 recipient were analyzed by flow cytometry (see A, right). The frequency of CD45.1+ LSK (LSK) and CD45.1+ LSKCD150 was calculated for 106 recipient (CD45.2+) cells and expressed as a percentage of the result obtained in the diaphysis in the same animal (n = 8, 2 independent experiments). (E) Analysis of cell viability 40 hours after lethal irradiation (time point where homing efficiency is being performed): BM cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and Annexin V in order to quantify the frequency of live (DAPI– Annexin V–), apoptotic (DAPI– Annexin V+), and necrotic (DAPI+ Annexin V+) cells (n = 4).
