Residues 39-56 of stem cell factor protein sequence are capable of stimulating the expansion of cord blood CD34⁺ cells

Background: Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC. Methods: Eleven polypeptides were synthesized, which were designed to cover the full-length ofrhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptideson human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometerwhereas CD34⁺ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture. Results: Of all polypeptides analyzed, only one, named P0, corresponding to the SCF proteinsequence at residues 39-56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34⁺ cells derived from human umbilical cord blood (UCB). Addition ofP0 increased the numbers of total mononucleated cells and CD34⁺ cells, by ∼2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansionassay. Conclusion: Residues 39-56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34⁺ cells. The peptide P0 is a potential candidate for furtherdevelopment as a synthetic substitute for rhSCF in laboratory and clinical applications.