HLF expression identifies repopulating cells in CD34⁺ CB cell cultures. (A) Fluorescence-activated cell sorting plots showing the sorting of HLF reporter–targeted population for transplantation. A pool of 8 CB units was split into 3 and processed as indicated. Cells were sorted for CD201⁺ at day 3 and nucleofected at day 4 using program DZ100, including siTP53 followed by transduction with multiplicity of infection 400 of rAAV6. (B) Summary of transplantation layout. Transplantation cohorts and cell doses are represented using the same color code as in panel A. (C) Human engraftment summary of transplant-recipient NSGS mice. Human bone marrow chimerism determined based on human CD45⁺ cells among total CD45⁺ (mouse and human) cells at short- (week 3), intermediate- (week 9), and long-term (week 16) posttransplantation time points plotted using the same color code as in panels A and B. Each recipient mouse is represented along the x-axis (NSGS ID). Recipients are arranged by descending average reconstitution across all time points. Recipients #25912, #25914, and #25916 were euthanized at week 10 posttransplantation to be used as donors for secondary transplantation (summarized in Figure 6). (D) Lineage proportion of transplant recipients. Bone marrow biopsies were analyzed and arranged as in panel C. Normalized proportions of B cells (CD19), myeloid cells (CD33), and T cells (CD3) within human CD45⁺ cells for each time point and recipient are color coded as indicated. (E) HR allele frequencies in pretransplanted cell populations. Top: ddPCR droplets are pregated based on FAM positivity; black droplets represent FAM⁺/HEX⁻ events indicative of untargeted alleles; red droplets (FAM⁺/HEX⁺) indicate targeted alleles, subsampled to 300 droplets per specimen. Bottom: quantification summary of HR frequencies calculated based on targeted/(untargeted + targeted) droplets. (F) ddPCR analysis of bone marrow biopsies at weeks 3, 9, and 16. Specimens are arranged as in panel C; ddPCR droplets are represented as in panel E, subsampled to 50 droplets per specimen and time point. (G) HR allele tracing summary. Summarized data representation of panels E and F. Dashed lines represent allele frequencies at the time of transplantation. Bars represent average HR allele frequencies from panel E with standard error bars; color codes as in panels A-C. One representative experiment of 2 independent experiments is summarized. PE, phycoerythrin.