PLAG1-S overexpression promotes self-renewal of long-term human HSCs. (A) Schematic of primary and secondary xenotransplantation in limiting dilution format. (B) Representative flow plots of human CD45⁺BFP⁺ multilineage (CD33⁺, CD19⁺) engraftment of primary recipient mice in injected femur. Percent human CD45⁺BFP⁺ engraftment in injected femur of primary recipient mice across multiple cell input doses. Dashed line indicates cutoff for calling engraftment, which was >0.5% human chimerism including both myeloid (CD45⁺BFP⁺CD33⁺) and lymphoid (CD45⁺BFP⁺CD19⁺) lineages. (C-D) Quantification of HSC frequency by ELDA⁴⁵ of injected femur of primary recipient mice. Shaded area under the curve represents 95% confidence interval of HSC frequency. (E) Percent human CD45⁺BFP⁺ engraftment in injected femur of secondary recipient mice across multiple cell input doses. Dashed line indicates cutoff for calling engraftment, which was the same as for primary mice. (F-G) Quantification of HSC frequency by ELDA of injected femur or uninjected bone marrow of secondary recipient mice and of in vivo expansion. Shaded area under the curve represents 95% confidence interval of HSC frequency. Total BFP⁺ cells within whole-body BM of primary mice were extrapolated, as previously,⁴⁷ based on femur and hind limb counts and proportional accounting from Colvin et al,¹⁰⁷ and in vivo expansion is measured as the fold difference of total BFP⁺ HSCs in donor mice relative to total day 0 HSCs initially transplanted into the 6 donor mice. Data are presented as average ± SEM unless otherwise indicated. Each point represents 1 mouse. See also supplemental Figure 3.