Competition from the host hematopoietic compartment is the primary barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Prostaglandin E2 (PGE2) has been shown to increase donor cell homing, cell survival, and engraftment after postnatal HCT through increased CXCR4 and Survivin expression. We have previously shown that pre-incubation with Diprotin A, a CD26 inhibitor, confers a homing and engraftment advantage for donor cells after IUHCT. We hypothesize that pre-incubation of donor cells with PGE2 alone, or in combination with Diprotin A, will provide a selective donor cell homing and competitive advantage and enhance long-term engraftment after IUHCT.
Balb/c pregnant mice underwent IUHCT with 1 × 107 B6GFP bone marrow (BM) cells, with or without pre-incubation with PGE2, Diprotin A, or PGE2 plus Diprotin A, by vitelline vein injection at E14. A 1μM per 1×106 BM cell concentration of dmPGE2, a long acting derivative, was used to treat the donor BM for two hours prior to IUHCT. In the combined therapy group, a 50 μM concentration of Diprotin A was used to treat the BM for an additional 15 minutes following PGE2 incubation. Homing to and engraftment in the fetal liver (FL) at early prenatal time points and frequency of donor cells in peripheral blood (PB) at 1 month and PB and BM at 4 months were assessed by flow cytometry. Analysis of donor cell cycling and cell survival was performed by in vitro BrdU cell labeling and anti-survivin and anti-caspase 3 antibody staining respectively.
Ex-vivo incubation of donor cells with PGE2 alone, Diprotin A, and PGE2 plus Diprotin A increased homing of allogeneic BM to the FL at 4 and 24 hours after IUHCT with the combined treatment group reaching statistical significance. Analysis of cell cycling of donor cells engrafted in the fetal liver at 96 hours after IUHCT demonstrated a 1.4 fold increase in the proportion of cells in the S and G2+M phases in PGE2 treated cells relative to non-treated BM controls (31.4% vs 21.2%) and a 1.2 fold increase over E18 FL controls (25.5%). A concurrent four fold increase in anti-survivin staining and a seven fold decrease in anti-caspase 3 staining were also seen in PGE2 treated cells engrafted in the fetal liver relative to non-PGE2 treated cells (anti-survivin: 19.8% vs. 4.75%. anti-caspase 3: 9.3% vs. 64.1%). Most importantly, a sustained increase in multi-lineage donor cell chimerism was observed (25–32%) in the PB and BM of recipients of PGE2 treated cells, as compared to controls, which demonstrated a decline from 13.4% to 4.9% over a 4 month period after IUHCT. Treatment with Diprotin A alone demonstrated a smaller increase in donor cell chimerism at 4 months after IUHCT. However, there was no statistical difference at 4 months between the PGE2 and PGE2 plus Diprotin A treatment groups (Figure 1).
PGE2 exposure results in markedly improved long-term engraftment of donor cells. This improvement is associated with increased cell cycling, increased survivin expression and reduced apoptosis suggesting that PGE2 exposure confers a competitive advantage to donor hematopoietic stem cells that persists over time by increasing cell cycling and survival. These increases likely explain the long-term advantage of PGE2 treatment. While CD26 blockade conferred an improvement in early homing kinetics, the combination of PGE2 plus CD26 blockade did not result in higher long term engraftment than PGE2 alone. We conclude that PGE2 pre-incubation is a promising method to achieve sustained increased donor cell engraftment after IUHCT.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.