Erythropoietin Blockade Improves Bone Marrow Homing of Umbilical Cord Blood Hematopoietic Stem Cells

Introduction Umbilical cord blood (UCB) is a promising modality for hematopoietic stem cell transplantation (HSCT) but is limited by cell yield from UCB units. This issue of cell dose can be addressed by improving homing of UCB stem/progenitor cells to the bone marrow (BM). Erythropoietin (EPO) receptor (EPOR) signaling blockade has arisen as a potential mechanism to improve BM homing and engraftment. Here we report on two novel EPOR inhibitors (URV2 and URV3) that show resolution of EPO mediated inhibition of cell migration in UCB CD34+ cells and in an EPOR expressing human erythroleukemia (HEL) cell line. URV2 and URV3 were identified by virtually screening compounds binding to EPOR. Methods Enriched UCB CD34+ cells were treated with URV2 or URV3 for 24 hours, with or without 100 ng/mL EPO, followed by 4 hour transmigration assay. HEL cells were treated with the same conditions, followed by 4 hour incubation at 37C and subsequent 10 hour transmigration assay. In transmigration assays, 1.0 mL of RPMI (0.5mL for UCB CD34+) with or without 125 ng/mL stromal derived factor 1 (SDF-1) was placed into the wells of a 12 (HEL cells) or 24 (UCB CD34+) well plate. An insert containing 12.0 um pores (8 um for UBC CD34+) was added to each well and 2 × 10^5 treated cells were seeded into the insert in 200uL of RPMI. Fold change was calculated by dividing percent migration in each condition (above migration in presence of EPO, without SDF-1) by migration in the presence of SDF-1 and absence of EPO. All experiments performed in duplicate at minimum; non-parametric ANOVA used for statistics. Results Mean HEL cell migration towards SDF-1 was reduced 0.22 fold in the presence of EPO (95% Confidence Interval (CI): -0.60-1.04), compared to no EPO treatment. Mean fold change in migration at 2.5, 5.0, 10, and 40uM URV2 was 0.25 (CI: -1.11-1.60), 0.47 (CI: -1.09-2.03), 1.30 (CI:0.71-1.87), and -0.10 (CI:1.37-1.17), respectively (p=0.026) (Fig. 1). Mean migration at 10uM URV2 was 11.99% (CI: 8.41%-15.57%). Fold change in migration at 2.5, 5.0, 10, and 40uM URV3 was 0.31 (CI:-0.87-1.49), 0.62 (CI:-0.45-1.70), 0.69 (CI:-0.71-2.08), and 2.5 (CI:-0.55-5.55), respectively (p=0.034) (Fig. 1). Mean migration at 40uM URV3 was 18.62% (CI: -14.47%-51.71%). In UCB CD34+ cells, fold change in migration with EPO and SDF-1 treatment, compared to SDF-1 treatment alone, was 0.69 (CI: 0.62-0.75). Migration in UCB CD34+ cells was recovered to 1.62 fold (CI: 0.35-2.90) over treatment with EPO and SDF-1 by addition of 40uM URV3 (p=0.06) (Fig. 2). Conclusions EPO has been shown as an inhibitor of cell migration in an in vitro model of BM homing. Novel blockade of EPOR with URV2 and URV3 restored migration in HEL cells comparable to migration without EPO. This trend was reproduced in UCB CD34+ cells with 40uM URV3 treatment. This improved UCB CD34+ cell homing may potentially reduce the incidence of complications arising from EPO-mediated suboptimal engraftment.