Conjugation of GSK3 inhibitor–loaded MLVs on B6-GFP BM-MNCs enhances long-term hematopoietic engraftment after IUHCT in Balb/c fetuses. (A) Experimental design: IUHCT of 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). (B) Fetal survival after 4 days post-IUHCT. There were no subsequent deaths. (C) Flow cytometry gating strategy for identifying CD45⁺ and GFP⁺ cells at P28 (light blue contour plots: CD45⁺, GFP⁻ controls). (D) Donor cell levels in blood at P28, P84, and P168 after IUHCT in 3 groups. At 4 weeks, chimerism was similar between animals in group 1 and survivors in group 2 (group 1, 15.5% ± 1.4% vs group 2, 10.5% ± 2.3%) but was more than 3 times higher in group 3 animals that received donor BM-MNCs carrying MLV-CHIR99021s (group 3, 52.9% ± 2.8%). (C-D) Engraftment levels in group 1 and 2 offspring were significantly reduced at 12 and 24 weeks of age compared with 4 weeks (group 1 at 12 weeks, 5.9% ± 0.7%; group 1 at 24 weeks, 3.4% ± 0.7%; group 2 at 12 weeks, 4.5% ± 1.0%; group 2 at 24 weeks, 2.9% ± 0.9%) but were maintained in group 3 animals (group 3 at 12 weeks, 48.3% ± 2.2%; group 3 at 24 weeks, 48.5% ± 3.1%). (E) Histograms for hematopoietic multilineage analysis using CD3, B220, Cd11b, and Gr1 antibodies in blood at 6 months (light blue histograms: CD45⁺, GFP⁺ [donor] or GFP⁻ [host] lineage-specific antibody-negative controls). (F) Multilineage reconstitution of all 3 groups at 6 months in blood compared with donor lineages (GFP control, normal nontransplanted donor). (B) *P < .05 group 2 vs groups 1 and 3; (D) *P < .05 group 1 at 4 weeks vs group 1 at 12 and 24 weeks; **P < .001 group 2 at 4 weeks vs group 2 at 12 and 24 weeks; ***P < .0001 vs groups 1 and 2 (all timepoints).