Figure 3.

In Vivo Migration of Human and Mouse HSPCs

CB MNCs were lineage depleted, stained with antibodies, and flow sorted to purify Lin⁻CD34⁺CD38⁻ (+/−) and Lin⁻CD34⁺CD38⁺ (+/+) populations. Cells were labeled and injected IV into NSG recipients.

(A–C) The displacement per hour (A), speed (B), and straightness (C) of (i) +/− and +/+ cells in μm in the calvaria at 16 hr and at days 2, 3, 4, and 6 post-transplantation. N = 10 recipients of cells from pooled CB units (two recipients for 16 hr, two recipients for day 2, three recipients of day 3, three recipients of day 4, and one recipient of day 6). Each recipient received cells from approximately six to nine CB donors. (ii) BM obtained from femurs and tibias of C57Bl/6 mice were lineage depleted, stained with Lin, SCA1, cKIT, CD150, and CD48 antibodies, and flow sorted to obtain LSK SLAM⁺ (Lin⁻, SCA1⁺, cKIT⁺, CD48⁻, CD150⁺) and LSK SLAM⁻ (Lin⁻, SCA1⁺, cKIT⁺, not CD48⁻, CD150+). The cells were labeled and injected IV into NSG recipients. The displacement per hour (A), speed (B), and straightness (C) of LSK SLAM⁺ and LSK SLAM⁻ in the calvaria at 16 hr is shown. N = 2 recipients at each time point of cells from pooled BM from approximately 10 C57Bl/6 donor mice for each recipient. (iii) CB MNCs were lineage depleted; stained with CD38, CD34, CD45RA, CD90, and CD49f antibodies (“CD49f”) or the cell dye Rhodamine123 (+/−/+/−/lo); and flow sorted to purify HSCs compared with HSPCs (CD34⁺/CD38⁻). Cells were labeled and injected IV into NSG recipients. Displacement per hour (A), speed (B), and straightness (C) of cells in the calvaria at 4 days after transplantation is shown. N = 3 recipients of CD49f⁺ cells, N = 2 recipients of +/−/+/−/lo cells, and N = 3 recipients of +/− cells of cells from approximately six to nine pooled CB units for each recipient. The +/− data in this figure are the same data from Figure 3A.

(D) The actual migration tracks for all +/− (green) and +/+ cells (red) from Figure 3A were overlaid on a 3D track plot (Wolfram Mathmatica) for day 3 (left) and day 4 (right). Total track length is displayed which ranges from 1–2 hr per cell.

(E) Sphericity for +/− cells at day 3 and day 4. Data obtained from the same cells in Figure 3A.