The Intravenous Route Optimizes Engraftment and Survival in the Murine Model of in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction

IUHCT has the potential to treat many congenital hematologic and immune disorders. It is a nonmyeloablative, nonimmunosuppressivetransplant approach that allows engraftment across immune barriers. Obtaining levels of engraftment necessary to treat target diseases remains a significant impediment to the clinical application of IUHCT. One barrier to consistent high-level engraftment is competition from host HSCs for limited niches at the time of transplant. The fetal liver (FL) is the hematopoietic organ in the mouse, large animals, and humans at the proposed time of IUHCT. Techniques that optimize donor cell delivery to the FL may enhance engraftment following IUHCT. In this respect, we sought to identify the route of injection that results in the least degree of mortality while promoting the highest level of engraftment. We compared the intravascular (IV), intraperitoneal (IP), and intrahepatic (IH) delivery of donor hematopoietic cells in a congenic murine model of IUHCT.  We hypothesized that the IV route would promote direct homing to the FL resulting in increased levels of postnatal peripheral blood (PB) chimerism, and allow for larger cell doses without deleterious effects on fetal survival. 

Methods

Five million C57Bl/6-GFP bone marrow mononuclear cells were suspended in a fixed volume of PBS (5 uL) and injected into embryonic day 14 (E14) C57Bl/6 fetuses via the IV, IP, or IH route. Flow cytometry and fluorescence stereomicroscopy (FSM) were used to assess donor cell homing to and engraftment of the FL at 4, 24 and 72 hours and the fetal spleen (FS) at 72 hours post IUHCT. Long-term postnatal PB engraftment was assessed monthly by flow cytometry. Fetal survival to E18 following increasing volumes of PBS injectate at E14 was compared between the different routes of injection. Statistical analysis was performed using ANOVA with Bonferronicorrection and FisherÕs Exact Test.

Results

The IV route led to significantly higher engraftment of the FL at 24 and 72 hours and the FS at 72 hours post IUHCT (Figure 1). FSM revealed important route-dependent differences in the patterns and kinetics of donor cell homing. IV injected donor cells robustly populated the FL in a homogenous manner within 4 hours of IUHCT. Most IP injected cells remained localized to the peritoneal cavity for over 24 hours post IUHCT with only modest engraftment of the FL. IH injected cells remained within the liver in a focal distribution with notable leaking into the perihepatic space beyond 24 hours post IUHCT. The increased efficiency with which donor cells homed to and engrafted the FL following IV IUHCT translated into superior and stable long-term donor cell engraftment (Figure 2). Fetal mice also demonstrated increased survival following higher injectate volumes administered via the IV route (Figure 3).  Tolerance to larger volumes permitted higher donor cell doses to be administered at a given concentration (1x10⁶ cells/mL), which translated into higher long-term donor cell engraftment (6 month chimerism, IV 5x10⁶ donor cells: 4.8 +/- 0.5% vs. IV 20x10⁶cells: 13.2 +/- 1.0%; p < 0.05).

Conclusions

In a congenic murine model of IUHCT, the IV route of injection results in increased levels of stable long-term donor cell engraftment compared to the IH and IP routes. Mechanistically, this is related to the increased efficiency of donor cell homing to the site of the hematopoietic niche at the time of the transplant. Furthermore, the IV route allows for larger volumes of injectate, and thus potentially higher cell doses, with reduced fetal loss. These observations have important implications in the translation of IUHCT technique to future clinical trials.