Figure 4.
GSK3 inhibitor released by MLVs increases engraftment after IUHCT by acting on HSCs/HPCs. (A) BM-HSCs were isolated and cultured over a 7-day period with (or without) an amount of CHI99021 similar to that carried by MLVs (per cell) added to the serum-free media. At the end of the culture period, ∼25% of the harvested cells were LSK, and this was not affected by the presence of CHIR99021 in the media. Flow cytometry gating strategy for sorting B6-GFP BM-HSCs (CD117+ and Sca1+ double-positive cells shown; light blue contour plots: CD117− and Sca1− double-negative controls). (B) Fold cell expansion after 7 days in culture in both groups (cultured without or with CHIR99021); total cells (left) and those only in the HSC compartment (right). Exposure of BM-HSCs to CHIR99021 over the 7-day culture period resulted in significant increase in the total number of both BM-MNCs and BM-HSCs. (C) Quantitative real-time polymerase chain reaction on the HSC group cultured with CHIR99021 demonstrated modulation of the Wnt (downregulation of Axin2 expression, upregulation of Ccnd1 expression), Notch (upregulation of Hes1 expression), and Hedgehog (upregulation Gli3 and Ptch1 expression) pathways, which have been shown to affect HSC function, proliferation kinetics, and repopulating capacity. (D) Hematopoietic colony-forming unit (CFU) assay; record of colony growth. (E) Hematopoietic CFU assays comparing CFU numbers in the different lineages in both cultured groups (without or with CHIR99021). BM-HSCs cultured in serum-free media with CHIR99021 had enhanced clonogenic potential evident in CFU (semisolid media) assays compared with untreated controls. (F) Experimental design: group 1, untreated BM-MNCs; group 2, BM-MNCs with cultured HSCs; group 3, BM-MNCs with HSCs cultured with GSK3 inhibitor; group 4, BM-MNCs in which fresh HSCs were conjugated with empty MLVs; and group 5, BM-MNCs in which fresh HSCs were conjugated with GSK3 inhibitor–loaded MLVs. Similar number of HSCs contained on the isolated fresh BM-MNCs was replaced by cultured HSCs. The HSC population is usually 0.1% of all BM-MNCs. Because the fetus received 107 BM-MNCs, this included 10 000 HSC, fresh, cultured, or MLV decorated. (G) Fetal survival after 4 days post-IUHCT in Balb/c fetuses in the 5 groups; there was no difference between groups in fetal survival (group 1, 69% vs group 2, 75% vs group 3, 72% vs group 4, 71% vs group 5, 67%). (H) Density plots of a sample of each group demonstrating the gating strategy for the identification of the CD45+ GFP+ population (light blue contour plots: CD45+, GFP− controls). (I) Donor cell (B6-GFP) levels in blood at P28, P84, and P168 after IUHCT in 5 groups. Levels of engraftment gradually decreased during the follow-up period (group 1 at 4 weeks, 14.3% ± 1.9%; group 1 at 12 weeks, 6.1% ± 0.8%; group 1 at 24 weeks, 3.9% ± 1.1%). Replacement of BM-HSCs from freshly isolated BM-MNCs with expanded BM-HSCs resulted in reduced blood engraftment at 4 weeks (group 4 at 4 weeks, 4.9% ± 0.6%), and only microchimerism was detected thereafter. Addition of CHIR99021 to the culture media of BM-HSCs prevented the latter drop in chimerism at 4 weeks (4 weeks: group 3, 12.9% ± 1.6%) but did not enhance it any further, resulting in an engraftment profile in blood similar to that observed in animals that received IUHCT of unprocessed/freshly isolated BM-MNCs (group 1). Conjugation of MLV-CHIR99021s on donor BM-HSCs resulted in an impressive increase in baseline engraftment levels post-IUHCT (4 weeks: group 5, 41.8% ± 3.7%), and this remained unchanged up to 24 weeks of recipient age (12 weeks: group 5, 41.4% ± 2.6%; 24 weeks: group 5, 45.4% ± 2.3%). The effect of MLV-CHIR99021s on engraftment was due to the gradual release and activity of the inhibitor, because conjugation of donor cells with empty particles did not reproduce the chimerism rise seen in group 5 animals (4 weeks: group 4, 19.2% ± 2.9%). (J) Multilineage reconstitution of all 5 groups at 6 months in blood compared with donor lineages did not demonstrate any differences between groups. (B,E) *P < .05 vs (-) CHIR99021; (I) *P < .05 groups 1-4: 4 weeks vs groups 1-4: 12 and 24 weeks and group 2: 4 weeks vs groups 1, 3, 4: 4 weeks; ***P < .0001 vs groups 1-4 (all timepoints).

GSK3 inhibitor released by MLVs increases engraftment after IUHCT by acting on HSCs/HPCs. (A) BM-HSCs were isolated and cultured over a 7-day period with (or without) an amount of CHI99021 similar to that carried by MLVs (per cell) added to the serum-free media. At the end of the culture period, ∼25% of the harvested cells were LSK, and this was not affected by the presence of CHIR99021 in the media. Flow cytometry gating strategy for sorting B6-GFP BM-HSCs (CD117+ and Sca1+ double-positive cells shown; light blue contour plots: CD117− and Sca1− double-negative controls). (B) Fold cell expansion after 7 days in culture in both groups (cultured without or with CHIR99021); total cells (left) and those only in the HSC compartment (right). Exposure of BM-HSCs to CHIR99021 over the 7-day culture period resulted in significant increase in the total number of both BM-MNCs and BM-HSCs. (C) Quantitative real-time polymerase chain reaction on the HSC group cultured with CHIR99021 demonstrated modulation of the Wnt (downregulation of Axin2 expression, upregulation of Ccnd1 expression), Notch (upregulation of Hes1 expression), and Hedgehog (upregulation Gli3 and Ptch1 expression) pathways, which have been shown to affect HSC function, proliferation kinetics, and repopulating capacity. (D) Hematopoietic colony-forming unit (CFU) assay; record of colony growth. (E) Hematopoietic CFU assays comparing CFU numbers in the different lineages in both cultured groups (without or with CHIR99021). BM-HSCs cultured in serum-free media with CHIR99021 had enhanced clonogenic potential evident in CFU (semisolid media) assays compared with untreated controls. (F) Experimental design: group 1, untreated BM-MNCs; group 2, BM-MNCs with cultured HSCs; group 3, BM-MNCs with HSCs cultured with GSK3 inhibitor; group 4, BM-MNCs in which fresh HSCs were conjugated with empty MLVs; and group 5, BM-MNCs in which fresh HSCs were conjugated with GSK3 inhibitor–loaded MLVs. Similar number of HSCs contained on the isolated fresh BM-MNCs was replaced by cultured HSCs. The HSC population is usually 0.1% of all BM-MNCs. Because the fetus received 107 BM-MNCs, this included 10 000 HSC, fresh, cultured, or MLV decorated. (G) Fetal survival after 4 days post-IUHCT in Balb/c fetuses in the 5 groups; there was no difference between groups in fetal survival (group 1, 69% vs group 2, 75% vs group 3, 72% vs group 4, 71% vs group 5, 67%). (H) Density plots of a sample of each group demonstrating the gating strategy for the identification of the CD45+ GFP+ population (light blue contour plots: CD45+, GFP− controls). (I) Donor cell (B6-GFP) levels in blood at P28, P84, and P168 after IUHCT in 5 groups. Levels of engraftment gradually decreased during the follow-up period (group 1 at 4 weeks, 14.3% ± 1.9%; group 1 at 12 weeks, 6.1% ± 0.8%; group 1 at 24 weeks, 3.9% ± 1.1%). Replacement of BM-HSCs from freshly isolated BM-MNCs with expanded BM-HSCs resulted in reduced blood engraftment at 4 weeks (group 4 at 4 weeks, 4.9% ± 0.6%), and only microchimerism was detected thereafter. Addition of CHIR99021 to the culture media of BM-HSCs prevented the latter drop in chimerism at 4 weeks (4 weeks: group 3, 12.9% ± 1.6%) but did not enhance it any further, resulting in an engraftment profile in blood similar to that observed in animals that received IUHCT of unprocessed/freshly isolated BM-MNCs (group 1). Conjugation of MLV-CHIR99021s on donor BM-HSCs resulted in an impressive increase in baseline engraftment levels post-IUHCT (4 weeks: group 5, 41.8% ± 3.7%), and this remained unchanged up to 24 weeks of recipient age (12 weeks: group 5, 41.4% ± 2.6%; 24 weeks: group 5, 45.4% ± 2.3%). The effect of MLV-CHIR99021s on engraftment was due to the gradual release and activity of the inhibitor, because conjugation of donor cells with empty particles did not reproduce the chimerism rise seen in group 5 animals (4 weeks: group 4, 19.2% ± 2.9%). (J) Multilineage reconstitution of all 5 groups at 6 months in blood compared with donor lineages did not demonstrate any differences between groups. (B,E) *P < .05 vs (-) CHIR99021; (I) *P < .05 groups 1-4: 4 weeks vs groups 1-4: 12 and 24 weeks and group 2: 4 weeks vs groups 1, 3, 4: 4 weeks; ***P < .0001 vs groups 1-4 (all timepoints).

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