A Pilot Expression And Purification Of Recombinant Human Interleukin3and Its Application In Expansion Of Hematopoetic Stem Cell

Objectives:To express and purify recombinant human interleukin3(rhIL-3), to raise a monoclonal antibody specifically against IL-3, and to functionally study rhIL-3and its antibody in stem cell expansion in vitro.Methods:rhIL-3was expressed and purified in a pilot scale. The recombinant cytokine was initially obtained in the form of inclusion body and it was refolded into the natural form through a graded dialysis process. Further purification was achieved by cation-exchange chromatography followed by gel filtration. To obtain monoclonal antibodies against IL-3, hybridoma cells were prepared from mice immunized with purified rhIL-3after which monoclonal antibody-secreting cell clones were screened and characterized by a combination of ELISA and Western blotting. To test the biological activity of rhIL-3, cord blood stem cells were cultured in a medium supplemented with various concentrations of rhIL-3in addition to stem cell factor (SCF,200ng/mL), fins like tyrosine kinase3ligand (Flt3,200ng/mL), and thrombopoietin (TPO,50ng/mL). The anti-IL-3antibody was added to the medium as additional control. Results were expressed as fold of total CD34+-positive cells after incubation.Results:rhIL-3protein was purified to homogeneity as detected by silver staining of SDS gel. The purified protein was stored at a concentration of0.52mg/mL. Three cell clones secreting IgGs specifically against IL-3were also obtained. Two clones belonged to the IgG1isotype and the third one belonged to the IgG2b isotype. Cell counting coupled with flow cytometry revealed that the optimal concentration of rhIL-3for CBSC expansion is15-20ng/mL (in the absence of TPO) or50ng/mL (in the presence of TPO). Moreover, two monoclonal antibodies from two cell clones were capable of neutralizing the activity of rhEL-3during in vitro cell expansion.Conclusions:Our combined studies suggest that rhIL-3obtained in this laboratory is highly active in supporting stem cell expansion in vitro. The purified cytokine and newly obtained monoclonal antibodies are valuable tools for stem cell expansion in preclinical and clinical studies.