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. 2015 Oct 8;5(5):690–701. doi: 10.1016/j.stemcr.2015.09.003

Figure 2.

Figure 2

Estimation of Stem Cell Frequency of Cells that Home to and Persist in the BM after Transplantation

(A) FACS analysis (top) of the CSFE+ CD34+CD38− before transplantation (bottom) FACS analysis of the CSFE+ cells extracted from the BM of NSG mice at day 4 post-transplantation.

(B) (left) Protocol to assess colony-forming ability of cells visible by microscopy; 300 Lin−CD34+CD38− (+/−) cells were plated directly into methylcellulose (before transplantation group) or stained with CFSE and injected IV into NSG recipient. After homing, mice were sacrificed; BM was extracted from femurs, tibias, and iliac crests and re-sorted to obtain CFSE+ cells; and these were plated into methylcellulose (after transplantation group). 75,000 cells from primary colonies were obtained after 14 days and re-plated to generate secondary colonies. (Right) Number of secondary colonies. N = 5 independent experiments from pooled CB units. Error bars represent ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

(C) (left) Protocol to assess stem cell frequency after transplantation. Lineage-depleted CB MNCs were stained with Lin, CD34, and CD38 antibodies and sorted by FACS to purify Lin−CD34+CD38− cells. Cells were injected intra-bone at limiting dose into sublethally irradiated recipients (before transplantation group) or stained with CFSE and injected IV into NSG recipient. After homing, mice were sacrificed, BM was extracted from femurs, tibias, and iliac crests and re-sorted by FACS to obtain CFSE+ cells and were injected intra-bone into the tibia at limiting dilution in sublethally irradiated recipients (after transplantation group). (Right) Engraftment at 12 weeks was assessed by flow cytometry for human CD45 multilineage (containing CD3, CD19, and CD33 cells) engraftment and scored positive if above 0.1%. ELDA software was used to calculate the frequency of repopulating cells. N = 48 mice (before and after cohort of mice used per condition: two mice each for 5, 20, and 40 cell doses, six mice each for 60 and 80 cell dose, from two independent experiments from approximately 9–12 pooled CB units).