Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications, and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here, we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF), thrombopoietin (TPO), insulin-like growth factor 2 (IGF-2), and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM "side population" (SP) cells, a highly enriched stem cell population, for 10 days resulted in an approximate 8-fold expansion of repopulating HSCs. Similar to freshly isolated HSCs, repopulating HSCs after culture were positive for the stem cell markers Sca-1, Kit, and CD31 and receptors for IGF-2. Surprisingly, prion protein and Tie-2, which are present on freshly isolated HSCs, were not on cultured HSCs. Two other HSC markers, Endoglin and Mpl, were expressed only on a portion of cultured HSCs. Therefore, the surface phenotype of ex vivo-expanded HSCs is different from that of freshly isolated HSCs, but this plasticity of surface phenotype does not significantly alter their repopulation capability.