Pseudoautocrine bioactivity of GSK3 inhibitor released by MLVs results in targeted augmentation of donor cell repopulating function. (A) Experimental design: IUHCT in Balb/c fetuses of a 1:1 mixture of B6GFP and B6-CD45.1 BM-MNCs with both populations unconjugated (group 1), with only B6GFP cells conjugated to GSK3 inhibitor–loaded MLVs (group 2), and with only B6-CD45.1 cells conjugated (group 3). (B) Fetal survival after 4 days post-IUHCT in 3 groups; there was no difference between groups (group 1, 71% vs group 2, 68% vs group 3, 69%). (C) Flow cytometry gating strategy for determining the percentage of both populations in blood; CD45.1⁺ (B6-CD45.1 cells) and GFP⁺ (B6-GFP cells) within the CD45 population; samples from the 3 groups (light blue contour plots: 45.1⁻ and 45.2⁻ double-negative controls; light blue histograms: GFP⁻ or CD45.1⁻ negative controls). (D) Mean percentage of GFP⁺ and CD45.1⁺ donor cells within groups 1, 2, and 3 in blood. Group 1 showed similar levels of engraftment in blood of recipients at 4 weeks of age (group 1: GFP⁺, 14.7% ± 2.2% vs CD45.1⁺, 12.1% ± 1.7%; GFP⁺/CD45.1⁺ ratio, 1.2 ± 1.7). Conjugation of MLV-CHIR99021s on either B6-GFP or B6-CD45.1 cells resulted in targeted enhancement of blood chimerism of the donor cell subpopulation carrying the inhibitor-loaded particles. In group 2, the percentage of GFP⁺ cells were significantly higher than CD45.1⁺ cells (group 2: GFP⁺, 33.2% ± 2.6% vs CD45.1⁺, 10.2% ± 1.4%; GFP⁺/CD45.1⁺ ratio, 3.3 ± 1.3). In group 3, the opposite was found (group 3: GFP⁺, 9.23% ± 2.1% vs CD45.1⁺, 33.3% ± 8.7%; GFP⁺/CD45.1⁺ ratio, 0.3 ± 1.1). (E-F) Multilineage analysis in GFP⁺ (E) and CD45.1⁺ (F) donor cells; no differences between groups in the proportion of lymphoid (CD3⁺, B220⁺) or myeloid (CD11b⁺, Gr-1⁺) cells in blood. (D) **P < .001 groups 2, 3: GFP⁺ vs CD45.1+; ***P < .0001 vs groups 1 and 2 or 1 and 3.