H/F-LVs transduce long-term reconstituting HSCs at levels approaching 100%. (A) Unstimulated hCD34⁺ cells were transduced with different H/F-LV preparations (H/F-1, H/F-2, H/F-3) encoding for a library of barcodes (32 bp long) and were injected into the livers of irradiated newborn NSG mice. Upon reconstitution, the bone marrow and spleen of these primary engrafted mice were analyzed for GFP⁺CD45⁺ cells in the BM and the spleen. An MOI of 5 was chosen. Upon 12 weeks of reconstitution, mice were euthanized and different populations from the BM (CD34⁺CD19^– early progenitors, CD34⁺CD19⁺ B-cell progenitors, CD13⁺ myeloid progenitors), spleen (CD19⁺, CD3⁺, CD14⁺ cells), and CD3⁺ thymocytes were isolated. The integrated bar codes in each of these subpopulations were determined and the shared bar codes between the different lineages are indicated in a Venn diagram. (B-E) Transduced hCD34⁺ cells were injected into the liver of irradiated newborn NSG mice. Upon reconstitution, the bone marrow and spleen of these primary engrafted mice (first transplantation) were analyzed for GFP⁺CD45⁺ cells in the BM and the spleen. Subsequently, the hCD34⁺ cells, isolated from the BM of these mice, were injected (following the same procedure as for the primary NSG engraftments) into secondary recipient mice. At 12 weeks from engraftment, these secondary NSG mice were analyzed for the percentage of transduced human cells (GFP⁺hCD45⁺) in the BM and spleen. Analysis of engraftment of transduced human cells (GFP⁺hCD45⁺) in the BM and spleen of primary (black bars) and secondary (white bars) transplanted mice reconstituted with hCD34⁺ cells (B) prestimulated with a cytokine cocktail (TPO + SCF + Flt3-L) or (C) unstimulated and transduced with H/F-LVs. Detailed phenotypic analysis of transduced human cells in the BM of secondary transplanted mice reconstituted with (D) prestimulated or (E) unstimulated H/F-LV transduced hCD34⁺ cells. Analysis of transduced human cells (GFP⁺) in the myeloid progenitors (CD13⁺), monocytes (CD14⁺), progenitor cells (CD34⁺), and B cells (CD19⁺) in secondary recipient mice.