Hematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized in clinical setting. Although umbilical cord blood (UCB) is a promising source of HSCs, insufficient number of HSCs in UCB limits their use, prompting need for ex vivo HSC amplification method.
HSCs emerge from hemogenic endothelium in the aorta-gonads-mesonephros (AGM) region, and then expand in liver. We previously showed that Delta-like 1 (Dlk1) positive hepatoblasts are the niche-like cells of HSCs in fetal liver. Dlk1 is a transmembrane protein and also secreted as soluble protein known as fetal antigen 1 (FA1). We hypothesized that hepatoblasts regulate HSC self-renewal via DLK1/FA1. Several bioactive peptides were generated from extracellular domain of Dlk1 and a bioactive peptide which has ability to expand HSCs was identified. UCB CD34+ cells were cultured with the bioactive peptide in serum-free medium containing a cytokine cocktail for 9 and 14 days. The number of CD34+CD38- cells were increased by bioactive peptide compared to control (9 days: 1.5 fold, 14 days: 2.5 fold). Transplantation of CD34+ cells cultured with bioactive peptide into immunodeficient NOG mice confirmed that the cultured cells possess long-term reconstitution ability. Moreover, we performed second transplantation and showed the self-renewal ability of the cultured cells, but not untreated control cells, suggesting that bioactive peptide has ability to expand HSCs and maintain its self-renewal.
To understand the mechanisms of HSC expansion by bioactive peptide, we investigated peptide binding proteins using biotin-conjugated bioactive peptide and identified PLEC and ERLIN2 proteins as interactors of bioactive peptide by LC-MS/MS and MASCOT analysis. PLEC knockdown UCB CD34+ cells cultured with bioactive peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of bioactive peptide. These results suggest that PLEC functions in HSC expansion promoted by bioactive peptide. In summary, we have identified a novel bioactive peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs.