A Structure-Function Study of the Activity of Stem Cell Factor in Stimulating the Expansion of Cord Blood CD34+ Cells

Stem cell factor is one of the most important growth factors for human hematopoietic stem cells (HSC). Recombinant human stem cell factor (rhSCF) can stimulate HSC expansion and regeneration in vitro, when it is used in combination with other cytokines like Flt-3L and TPO. However, the specific structural region(s) of the rhSCF protein that are critical for its function in HSC expansion are still unknown. Few studies have addressed this problem, to date. We have recently reported the production of a novel monoclonal antibody (named 23C8) against rhSCF, and the demonstration that 23C8 could inhibit the ability of rhSCF to enhance HSC expansion. Here, we report the identification of a short polypeptide from rhSCF that contains the epitope for binding to 23C8, and, like the full-length rhSCF, is able to stimulate the expansion of umbilical cord blood (UCB)-derived CD34+ cells.

Twelve short polypeptides were designed and synthesized, which cover the full length of rhSCF, with 3-5 amino acids overlaps. 23C8 was collected from hybridoma cell culture medium and further purified using protein G affinity chromatography. ELISA was used to identify the polypeptide(s) that positively react with 23C8 among all the synthesized polypeptides. In addition, the effects of the synthetic polypeptides on human HSC expansion capacity were evaluated by supplementing the cell culture medium with 100 ng/ml of a given polypeptide. Total cell number and CD34+ cell number of each group were monitored on day 6. Our novel anti-SCF monoclonal antibody (23C8) partially blocked SCF’s function in human UCB CD34+ cell expansion. Of all the polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 40 to 57, was recognized by 23C8 during ELISA. P0, like the full-length rhSCF, enhanced expansion of CD34+ cells derived from human UCB. P0 addition increased the numbers of total nucleated cells and CD34+ cells by 10.58±0.86 and 4.63±0.43 folds, respectively. For comparison, the extents of increases in cell numbers in the vehicle control group was 3.15±0.99 fold (total nucleated cells) and 1.07±0.11 fold (CD34+ cells), respectively. Residues 40-57 of hrSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells in vitro. The short P0 peptide is a potential candidate for development as a synthetic substitute for rhSCF in clinic applications.