The Use of Eicosanoids to Enhance Donor Cell Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE₂) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment.

Methods

Ten million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2K^b) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2K^d) via the vitelline vein. Donor cells were treated with vehicle, PGE₂, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE₂ and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM.

Results

PGE₂ pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p < 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE₂ treatment. PGE₂ treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p<0.05) and decreased donor cell anti-caspase 3 staining (9.3±1.2% vs. 64.1±10.6%; p<0.05) at 96 hours post-IUHCT. These findings of increased FL/FS engraftment and increased survival at 72 to 96 hours post IUHCT following donor cell treatment with PGE₂ translated into significantly increased long-term donor cell engraftment up to 6 months of age. Similar to PGE₂, ex vivo treatment of donor cells with 11,12-EET or 14,15-EET resulted in enhanced long-term allogeneic donor cell engraftment (Figure 1). Balanced multilineage engraftment was seen for all treatments suggesting enhanced engraftment at the level of the HSC. No deleterious effects were observed on murine growth or overall health.

Conclusions

The ex vivo treatment of donor cells with eicosanoids including PGE₂, 11,12-EET, and 14,15 EET prior to IUHCT results in enhanced long-term multilineage allogeneic donor cell engraftment. Detailed studies of early homing to fetal hematopoietic organs and survival at 96 hours following IUHCT of PGE₂ treated donor cells suggest the mechanism of enhanced engraftment is mainly do to a pro-survival effect of PGE₂ and related eicosanoids. This work represents a novel application of these eicosanoids in an allogeneic model of IUHCT and highlights their potential to assist in future clinical applications of IUHCT.

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