HLF reporter labels human HSCs with extensive self-renewal capacity. (A) Fluorescence-activated cell sorting plots of CD34⁺/HLF-ZsG⁺ population. Representative bone marrow biopsies of reporter-negative (sgHLF/rAAV6 HLF-ZE targeted, ZsG⁻ sorted, left) and reporter-positive (sgHLF/rAAV6 HLF-ZE targeted, ZsG⁺ sorted, right) primary recipients, gated on human CD45⁺. (B) Summary of CD34⁺/HLF-ZsG⁺ population. Population overview of all primary recipients, pregated on human CD45⁺; recipient mice are arranged according to engraftment levels as in Figure 5C. (C) Strategy for secondary transplantation. The bone marrow of 3 primary recipients (sgHLF/rAAV6 HLF-ZE targeted, ZsG⁺ sorted cohort) was pooled and magnetically enriched for human CD34 expression. Reporter-expressing (ZsG⁺) and -nonexpressing cells (ZsG⁻) with comparable levels of CD34 expression were sorted for transplantation. Intrahepatic transplantation into newborn NSGS recipients as outlined. Corresponding cell doses of HLF-ZsG⁺ (n = 10) and HLF-ZsG⁻ (n = 7) were transplanted. (D) Human engraftment summary of secondary recipients. Human chimerism in indicated tissues was determined based on human CD45-expressing cells among total (mouse and human) CD45⁺ cells at short- (week 5; blood), intermediate- (week 9; marrow), and long-term (week 16; marrow and spleen) time points posttransplantation. The dashed line represents the 0.1% mark used as the cutoff for engraftment positivity. Significance was calculated by unpaired 1-sided (alternative indicates greater) Wilcoxon test and is provided as a P value for a given comparison. (E) Lineage output of engrafted human cells. Positive specimens from panel D are shown and color coded for B cells (CD19⁺), myeloid cells (CD33⁺), and T cells (CD3). Normalized for lineage proportions within human CD45⁺ cells. Samples with <0.1% of human chimerism are designated negative (neg.). APC, allophycocyanin.