Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells

Objective. In an attempt to maintain and expand human stem cells, many inve stigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, ho wever, the safety of the delivery of grafts expanded in culture using xenog eneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human pri mitive progenitors and transplantable stem cells. Material and Methods. Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (T PO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogen ic progenitors and severe combined immunodeficient disorder (SCID) mouse-re constituting cells (SRC), Results. In the presence of TPO, FL, and SCF, marrow stromal cells supporte d more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-for ming units in culture after 2 and 4 weeks of incubation, respectively. In a ddition, cobblestone area-forming cells were expanded more than 18- and 60- fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay d emonstrated augmented engraftment by cultured cells. Conclusion. This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors. (C) 2001 International Society for Experimental Hematology. Publis hed by Elsevier Science Inc.