Long-term engraftment in BM after IUHCT of B6-GFP BM-MNCs conjugated with GSK3 inhibitor–loaded MLVs in Balb/c fetuses. (A) Flow cytometry gating strategy for identifying CD45⁺ and GFP⁺ cells in BM in 3 groups: BM-MNCs alone (group 1), BM-MNCs associated with a bolus of GSK3 inhibitor (group 2), and BM-MNCs conjugated with GSK3 inhibitor MLVs (group 3) (light blue contour plots: CD45⁺, GFP⁻ controls). (B) Percentage of CD45⁺ GFP⁺ donor cells on host BM at 6 months in the 3 groups. Group 1 and 2 animals had similar levels of donor cell engraftment (group 1, 1.6% ± 0.4% vs group 2, 2.1% ± 0.9%), whereas levels in group 3 animals were almost 25 times higher (group 3, 50.4% ± 2.6%). (C) Flow cytometry gating strategy for hematopoietic multilineage analysis using CD3, B220, Cd11b, and Gr1 antibodies in BM at 6 months (light blue histograms: lineage-specific antibody-negative controls). (D) Multilineage reconstitution of all 3 groups at 6 months in BM compared with donor lineages demonstrated no differences between groups. (E) BM histology with immunohistochemistry for GFP (brown) at 6 months in all 3 groups and Balb/c and GFP control mice (magnification: ×40; scale bar 100 μm; stain: diaminobenzidine [brown] and hematoxylin [blue]). (B) ***P < .0001 vs groups 1 and 2.