Effect of targeted pseudoautocrine bioactivity of GSK3 inhibitor released by MLVs on donor HSCs/HPCs. (A) Flow cytometry gating strategy for determining the HPC/HSC (Lin⁻) and HSC-containing compartments of donor cells found in BM of IUHCT recipients (Balb/c fetuses); samples from the 3 groups (light blue contour plots: 45.1⁻ and 45.2⁻ double-negative controls or CD117⁻ and Sca1⁻ double-negative controls; light blue histograms: Lin⁻, GFP⁻, or CD45.1⁻ negative controls). (B) Proportion of donor Lin⁻ cells (expressed as a percentage of the sum of CD45.1⁺ and CD45.2⁺ BM-MNCs) was 2.8% ± 0.2% in group 1, and no differences were observed among experimental groups (Lin⁻: group 2, 3.1% ± 0.3% vs group 3, 3.3% ± 0.4%). (C) Absolute number of donor Lin⁻ cells (per hind limb long bone); significant increase in hematopoietic progenitor number only in the donor cell subpopulation subjected to targeted GSK3 inhibition (group 1: Lin⁻, GFP⁺, 2.5 × 10³ ± 0.4 × 10³ vs Lin⁻, CD45.1⁺, 4.8 × 10³ ± 0.5 × 10³; group 2: Lin⁻, GFP⁺, 16.7 × 10³ ± 1.9 × 10³ vs Lin⁻, CD45.1⁺, 5.7 × 10³ ± 1.5 × 10³; group 3: Lin⁻, GFP⁺, 2.5 × 10³ ± 0.3 × 10³ vs Lin⁻, CD45.1⁺, 21.2 × 10³ ± 5.2 × 10³). (D) Ratio of GFP⁺/CD45.1⁺ Lin⁻ cells. (E) Proportion of donor primitive LSK cells (expressed as a percentage of the Lin⁻ cells in the sum of CD45.1⁺ and CD45.2⁺ BM-MNCs); no differences were observed among experimental groups (LSK: group 1, 11.2% ± 0.6% vs group 2, 9.8% ± 1.5% vs group 3, 8.8% ± 1.4%). (F) Absolute number of LSK cells (per hind limb long bone); significant increase only in the donor cell subpopulation carrying MLV-CHIR99021s (group 1: LSK, GFP⁺, 3.1 × 10² ± 0.6 × 10² vs LSK, CD45.1⁺, 4.9 × 10² ± 0.7 × 10² CD45.1⁺ cells; group 2: LSK, GFP⁺, 16.1 × 10² ± 1.6 × 10² vs LSK, CD45.1⁺, 6.1 × 10² ± 1.9 × 10²; group 3: LSK, GFP⁺, 2.4 × 10² vs LSK, CD45.1⁺, 18.8 × 10² ± 6.2 × 10². (G) Ratio of GFP⁺/CD45.1⁺ LSK cells. (C) *P < .05 group 2: Lin⁻, GFP⁺ vs Lin⁻, CD45.1⁺, **P < .001 group 3: Lin⁻, GFP⁺ vs Lin⁻, CD45.1⁺; (D) ***P < .0001 vs groups 1 and 2 or 1 and 3; (F) *P < .05 group 2: GFP⁺ vs CD45.1⁺, **P < .001 group 3: GFP⁺ vs CD45.1⁺; (G) **P < .001 vs groups 1 and 2 or 1 and 3.