Fig 2: Schematic diagram of generating functional T lymphocytes by reprogramming in vivo. The recombinant Hoxb5 vector or GFP empty vector control was transduced into Plat-E cells to produce viruses. Pro/pre-B cells (Ter119–Mac1–CD3–CD4–CD8–B220+CD19+CD93+IgM–) were sorted from the bone marrow and then cultured in medium (15%FBS, 100 mM GlutaMAX, 10-4 M ß-ME, 10 ng/ml mSCF, 10 ng/ml Flt3L and 10 ng/ml IL7) for 12–16 h before virus transduction. Pro/pre-B cells (1 × 106 per ml) were transduced with the mixed viruses with equal pre-adjusted titers. The pro/pre-B cells transduced with retro-Hoxb5 virus were collected for transfection efficiency analysis and then were transplanted into sublethally irradiated syngeneic recipient mice via orbital veins. Four weeks after transplantation, cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry.

Fig 3: Flow cytometry analysis of the purity of sorted Ter119–Mac1–CD3–CD4–CD8–B220+CD19+CD93+IgM–pro/pre-B cells. Bone marrow nucleated cells were first incubated with biotin-conjugated anti-B220 antibody and then enriched by streptavidin microbeads using AutoMACS Pro. The pro/pre-B cells were further sorted from the B220 enriched cells by defined markers. Flow cytometry analysis confirmed the purities of B220 enriched cells (top) and sorted pro/pre-B cells (bottom).

Fig 4: AAV-Transduced Hematopoietic Stem Cells Exhibit Long-Term Reconstitution Potential following Secondary Transplantation(A) Percentage of donor chimerism among live peripheral blood cells in secondary recipients at monthly intervals post-transplantation. Data points represent mean ± SD. n = 6–9 secondary recipients per group.(B) Frequency of secondary recipients with tdTomato+ multilineage engraftment within indicated peripheral blood lineages at 16 weeks post-transplant. tdTomato+ engraftment was determined using the same criteria as in Figure 3E.(C–F) Frequency of tdTomato+ cells among live CD45.2+ peripheral blood (C) T cells, (D) B cells, (E) monocytes, and (F) neutrophils in secondary recipients at 16 weeks post-transplantation. For each lineage, only recipients exhibiting >1% CD45.2+ cells within that lineage are shown. Individual data points are shown overlaid with mean ± SD. n = 6–9 mice per group.(G) Percentage of tdTomato+ HSCs among CD45.2+ HSCs in secondary recipients at 4–5 months post-transplantation. Individual data points are shown overlaid with mean ± SD. n = 6–9 secondary recipients per group.vg, viral genomes; Anc80, Anc80L65.See also Figures S5 and S6 and Table S1.

Fig 5: Systemic Injection of AAV-Cre Transduces Functional Hematopoietic Stem Cells(A) Experimental design. AAVs encoding Cre recombinase were injected into mdx;Ai9 mice carrying the Rosa26-LSL-tdTomato reporter. i.v., intravenous; LSL, LoxP-STOP-LoxP; HSPCs, hematopoietic stem and progenitor cells.(B) HSC gating strategy (Lin-Sca-1+c-Kit+CD48-CD150+) from bone marrow (BM) and representative flow cytometric analysis of tdTomato expression within HSCs. LSK: Lin-Sca-1+c-Kit+.(C) Frequency of HSCs expressing tdTomato. Data points from individual mice are overlaid with mean ± SD. n = 4 mice injected with each AAV serotype per group, n = 3 vehicle-injected mice per group.(D) Percentage of donor chimerism among live peripheral blood cells of primary recipients at monthly intervals post-transplantation. Data points represent mean ± SD. n = 8 primary recipients per group.(E) Frequency of primary recipients with tdTomato+ multilineage engraftment within the indicated peripheral blood lineages at 16 weeks post-transplant. For each lineage, tdTomato+ engraftment was scored based on satisfaction of two criteria: >1% CD45.2+ cells and >1% tdTomato+ among CD45.2+ cells.(F–I) Frequency of tdTomato+ cells among live CD45.2+ peripheral blood (F) T cells, (G) B cells, (H) monocytes, and (I) neutrophils in primary recipients at 16 weeks post-transplantation. For each lineage, only recipients exhibiting >1% CD45.2+ cells within that lineage are shown. Individual data points are shown overlaid with mean ± SD. n = 6–8 primary recipients per group.(J) Percentage of tdTomato+ HSCs among CD45.2+ HSCs in primary recipients at 6 months post-transplantation Individual data points are shown overlaid with mean ± SD. n = 6–8 primary recipients per group.vg, viral genomes; Anc80, Anc80L65.See also Figures S3–S6 and Tables S1 and S2.