LNK depletion improves engraftment defects of FA-like HSCs in xenotransplant. (A) Experimental scheme of xenotransplantation assay. CB-derived CD34⁺ cells were first transduced with lentivirus-expressing GFP and either shLuc or shFANCD2 to establish FA-like HSPCs. Subsequently, the cells were transduced with lentivirus-expressing mCherry and either shLuc or shLNK #1 and transplanted into NBSGW recipients. The PB was analyzed every 4 weeks for 16 weeks, and after 16 to 22 weeks, the BMs and spleens were analyzed. (B) Total human chimerism (percent hCD45⁺) in xenotransplanted mice in the PB at 16 weeks, BM and spleen (Spl) is shown. * = P < .05, calculated using 2-way ANOVA followed by Dunnett’s multiple comparisons to nontransduced control. (C) Mean percent hCD45⁺GFP⁺mCherry⁺ chimerism in the PB at 16 weeks; BM and Spl are shown. (D) Mean GFP/mCherry transduction rates at time of transplant from 4 different CBs are shown. (E) Chimerism of each transduced population (GFP⁺mCherry⁺, GFP⁺mCherry⁻, GFP^-mCherry⁺, and GFP⁻mCherry⁻) in the BM or spleen are shown as stacked means (left panel). The right panel illustrates the predicted reduction or expansion of each transduced cell population upon FANCD2 or LNK depletion. (F) Mean percent GFP⁺mCherry⁺ within each lineage of the engrafted hCD45⁺ cells in the BM (T cells [T] = CD3⁺, B cells [B] = CD19⁺, myeloid [Mye] = CD33⁺, erythroid progenitors [Ery] = CD235a⁺, megakaryocytes [Mk] = CD41a⁺). (G) Mean percent GFP⁺mCherry⁺ in the engrafted CD34⁺ HSPC compartment in the BM. In all relevant panels, each symbol represents an individual mouse; bars indicate mean values; error bars indicate plus or minus SEM. *P < .05; **P < .01; ***P < .001 as determined by 2-way ANOVA followed by Tukey’s multiple comparisons. N = 12 shLuc/shLuc, n = 15 shFANCD2/shLuc, n = 15 shFANCD2/shLNK, and n = 5 for nontransduced transplanted mice. The data are pooled from 4 different biological replicate CBs.