Analysis of HSC pool in mice transplanted with old and young cells. (A) Shows experimental setup of LSK48⁻150⁺ cells that were purified from CD45 congenic young (4 months) and old (24 months) donor mice, mixed in 1:2 ratio and transduced with the barcoded vector library. There were 20 500 transduced cells transplanted simultaneously with W41 or previously transplanted B6 cells into 2 cohorts of 4 lethally irradiated B6 mice. There were 6 mice sacrificed 6 months post-transplantation, and 2 additional mice sacrificed at 8 months after transplantation. GFP⁺ LSK48⁻150⁺ cells of young and old origin were single-cell sorted in 96-well plates and expanded in liquid culture in presence of cytokines for subsequent barcode analysis. (B) Contribution from young (white bars) and old (gray bars) to different cell populations before (starting) and after transplantation. (C) Number of uniquely barcoded clones detected in expanded colonies of young and old LSK48⁻150⁺ cells. Lines connect data points derived from the same mice. The number of clones detected within the old compartment was significantly higher than the number of clones within the young population (P = .0011, paired two-sided t test). (D) Contribution of individual young and old LSK48⁻150⁺ HSCs to the stem cell compartment. Horizontal lines indicate mean values. Young LSK48⁻150+ cells produced larger clones than old LSK48⁻150⁺ cells (P < .0001, two-tailed Mann Whitney nonparametric U test).