Human Cell Expressed Cytokines for Expansion of CD34+ Hematpoietic Stem Cells.

Abstract

Many cytokines and growth factors are heavily glycosylated, with up to 75% of their mass consisting of carbohydrate moieties. Glycosylation is important for secretion, solubility, resistance to proteolysis, immunogenicity, biological recognition, biological activity, in vivo stability and clearance of glycoproteins including cytokines and growth factors. This study was designed to compare the ability of human cell expressed cytokines to induce CD34+ hematopoietic stem cell expansion with human cytokines expressed in E. coli. CD34+ stem cells were cultured with either human cell expressed G-CSF and stem cell factor (SCF), or with human G-CSF and SCF expressed in E. coli. CD34+ stem cell expansion was determined by counting total nucleated cells (TNC), and colony forming unit (CFU) potential of the expanded cells was determined by colony number, size and cell morphology. During early cell proliferation and expansion, the TNC count of cells treated with the human cell expressed cytokines was markedly higher than cells treated with E. coli expressed human cytokines. In CFU assays, the size and mean number of colonies were greater with the human expressed G-CSF and SCF cytokine combination. In addition, the proportion of colonies with macrophage-like morphology was greater. Human cell expressed cytokines also induced greater myeloid differentiation as indicated by higher CD33 expression compared to cytokines expressed in E. coli.We are currently investigating the ability of human cell expressed G-CSF/SCF to activate signaling pathways including the MEK/ERK and STAT pathways, which are known to be involved in G-CSF mediated proliferation and differentiation. Differences in receptor-ligand binding using ligands from various expression systems are also being examined.The ability of human cell expressed cytokines to induce greater in vitro differentiation and proliferation of hematopoietic stem cells may translate to better ex vivo expansion protocols.