1. The Intravenous Route of Injection Optimizes Engraftment and Survival in the Murine Model of In Utero Hematopoietic Cell Transplantation 
Matthew M. Boelig, Aimee G. Kim, John D. Stratigis, Lauren E. McClain, Haiying Li, Alan W. Flake, William H. Peranteau 
Biology of Blood and Marrow Transplantation 
Volume 22, Issue 6, Pages (June 2016)
DOI: /j.bbmt
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Biology of Blood and Marrow Transplantation 2016 22, 991-999DOI: (10
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 1
Early kinetics of donor cell homing and engraftment after IUHCT. Embryonic day 14 B6 mice were injected with 5 × 106 B6GFP BM cells. Total donor cell homing and engraftment was assessed in the FL at 4, 24, and 72 hours after IUHCT (A) and in the FS at 72 hours after IUHCT (B). Donor HSC homing to the FL as a function of total FL CD45+ cells (C) and the proportion of donor CD45+ cells in the FL (D) were assessed at 24 and 72 hours after IUHCT. Survival of donor cells in the FL at 24 and 72 hours after IUHCT was assessed by Annexin V staining (E). For (A), the number of injected fetuses analyzed (n) at 4, 24, and 72 hours after IUHCT = 6, 26, 16 (i.v.); 16, 29, 12 (i.p.); 14, 8, 8 (i.h.), respectively. For (B), n = 6 (i.v.), 5 (i.p.), 8 (i.h.). ★P < .05; i.v. versus i.p. and i.h. For (C), (D) and (E), n = 9 (i.v.), 13 (i.p.), and 12 (i.h.) at 24 hours and 6 (i.v.), 8 (i.p.), and 12 (i.h.) at 72 hours.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 2
Fluorescence stereomicroscopy (FSM) images of fetal hematopoietic organs after IUHCT with GFP+ donor cells via the i.v., i.p., and i.h. routes. Embryonic day 14 B6 mice were injected with 5 × 106 B6GFP BM cells via either the i.v., i.p. or i.h. route. FLs (A) and whole abdomens with organs in situ (C) were assessed at 4, 24 and 72 hours after IUHCT and FSs were assessed at 72 hours after IUHCT (B) for the presence of B6GFP donor cells. SI indicates, small intestine; white arrow, intraperitoneal donor cell clump; white star, intrahepatic injection site. Representative images are displayed. All images taken at an exposure time of 3700 ms and magnification as indicated.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 2
Fluorescence stereomicroscopy (FSM) images of fetal hematopoietic organs after IUHCT with GFP+ donor cells via the i.v., i.p., and i.h. routes. Embryonic day 14 B6 mice were injected with 5 × 106 B6GFP BM cells via either the i.v., i.p. or i.h. route. FLs (A) and whole abdomens with organs in situ (C) were assessed at 4, 24 and 72 hours after IUHCT and FSs were assessed at 72 hours after IUHCT (B) for the presence of B6GFP donor cells. SI indicates, small intestine; white arrow, intraperitoneal donor cell clump; white star, intrahepatic injection site. Representative images are displayed. All images taken at an exposure time of 3700 ms and magnification as indicated.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 3
Postnatal engraftment after IUHCT via i.v., i.p., and i.h. routes. Embryonic day 14 B6 mice were injected with 5 × 106 B6GFP BM cells. PB donor cell engraftment was analyzed at 1, 2, 3, and 6 months of age (A). Engraftment levels were further analyzed to determine the percentage increase in chimerism achieved by the i.v. route of injection compared with that by the i.p. and i.h. routes at the above time points (B). ∗P < .05; i.v. versus i.p. and/or i.v. versus i.h., as indicated.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 4
Multilineage engraftment after IUHCT via the i.v., i.p., and i.h. routes. Embryonic day 14 B6 mice were injected with 5 × 106 B6GFP BM cells. PB was analyzed at 6 months of age for the presence of GFP + cells that expressed CD3 (T cells), B220 (B cells), Gr-1 (granulocytes), and CD11b (macrophages) to assess for donor cell multilineage engraftment. There were no significant differences in multilineage chimerism levels between the 3 injection routes.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

8. Figure 5
Fetal survival trends with escalation of IUHCT injectate volumes. Embryonic day 14 B6 fetuses underwent in utero injection with increasing volumes of sterile PBS via the i.v., i.p., and i.h. routes. Fetuses were assessed before delivery (embryonic day 18.5) for viability (spontaneous movements, gross appearance, cardiac activity). The i.v. route was associated with significantly increased survival to embryonic day 18.5 at injectate volumes > 15 μL (versus i.h.) and >20 μL (versus i.p.). ∗P < .05, i.v. versus i.p.; ψP < .05, i.v. versus i.h.; ψP < .05, i.p. versus i.h.. The number of fetuses (n) assessed at the 5, 10, 15, 20, 25, 30, and 35-μL injection volumes respectively were – i.v.: 11, 10, 15, 27, 10, 30, 23; i.p.: 17, 13, 17, 30, 18, 26, 23; i.h.: 15, 19, 24, 35, 28, 32, 37.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

9. Figure 6
Enhanced engraftment after increased donor cell doses provided by i.v. IUHCT. Embryonic day 14 B6 fetuses were injected with 5 × 106 or 20 × 106 B6GFP BM cells at a concentration of 1 × 106 cells/mL via the i.v. route. PB donor cell engraftment levels were assessed at 1, 2, 3, and 6 months of age. Donor cell chimerism was significantly higher at all time points following injection of the higher donor cell dose. ∗P < .05; 20 × 106 versus 5 × 106 donor BM cells.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

10. Supplemental Figure S1
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

11. Supplemental Figure S2
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

12. Supplemental Figure S3
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions

13. Supplemental Figure S4
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2016 The American Society for Blood and Marrow Transplantation Terms and Conditions