Hematopoietic stem cell expansion under serum-free and cytokine-limited conditions using primary endothelial cells transfected with the adenoviral E4-ORF1 gene.

摘要

Research over the past 100 years has shown that blood cells develop in close proximity to the vascular system during embryogenesis and that hematopoietic reconstitution following bone marrow ablative therapy is dependent on rapid regeneration of the bone marrow vascular niche (BMVN). Studies to determine the role of the BMVN in regulating the recovery of long-term repopulating HSCs (LT-HSCs) have however, been hampered by a lack of models that provide faithful conditions with which to observe self-renewal in-vitro. Here we describe a novel culture system in which the introduction of a single adenoviral peptide cloned from early region 4, open reading frame 1 (E4-ORF1) allows for the culture of primary endothelial cells (PECs) under serum-free and growth factor-free conditions. Using this system we have been able to interrogate the role of PECs in HSC self-renewal and demonstrate conclusively, for the first time, that endothelium contributes directly to LT-HSC expansion and self-renewal.;Co-culture of murine Sca1+cKit+Lin - (KLS) cells with PECs resulted in a robust expansion (700 fold) of phenotypic HSCs, which sustained continued expansion beyond 2 months of culture. The expanded hematopoietic cells were able to reconstitute lethally irradiated recipients and gave rise to long-term engraftment as determined by serial transplantation into secondary recipients.;With this technology we have been able to demonstrate that E4-ORF1-transduced primary PECs expand TNR.GFP+ HSCs through a Jagged/Notch-dependent mechanism. Inhibiting Notch signaling through the addition of gamma-secretase inhibitor reduces the number of LT-HSCs and forces maturation and lineage-specific differentiation. These data present a novel tool for studying the role of BMVN in HSC expansion and provide some initial insights into possible mechanisms governing HSC self-renewal.