OBJECTIVETo evaluate the effects of endothelial cell- targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cell (CB HSPCs).

METHODSRecombinant hD1R protein was first induced and purified. Human cord blood CD34⁺ cells were co-cultured on human umbilical vein endothelial cells (HUVECs) supplemented with a cocktail containing 5 types of human cytokines including TPO, SCF, FL, IL-6, IL-3 (5GF) and soluble hD1R. The expansion of CD34⁺ cells was tested under different culture conditions including PBS group (PBS replaces HUVEC), hD1R group, sup group (HUVEC supernatant replaces HUVEC), fix group (fixed HUVEC replaces HUVEC), Day 0 group (Control). Cell cycle and apoptosis of cultured cells were also analyzed. Their progeny expanded in PBS or hD1R group were transplanted into sublethally irradiated NOD/SCID mice. The percentages of human CD45⁺ (hCD45⁺) cells in the marrow of recipient mice were determined by FACS 12 weeks later.

RESULTShD1R induced more expansion in the total number of CD34⁺ cells cocultured with HUVECs plus 5GF, which was 87.50-fold increase compared to the Day 0 group, and 7.98-fold increase than that of PBS group. FACS analysis also showed that the percentage of CD34⁺ cells was 77.0% in G0/G1 phase in the hD1R group, which indicated that hD1R enhanced HSPCs expansion and inhibited apoptosis. Moreover, hD1R significantly promoted human HSPC engraftment after BM transplantation in irradiated mice.

CONCLUSIONThe Notch-mediated ex vivo expansion system has been established and hD1R promoted expansion and engraftment of human CB HSPCs, which provided the evidence for further clinical application.