Ex vivoExpansion Potential of Murine Hematopoietic Stem Cells: A Rare Property Only Partially Predicted by Phenotype

Abstract

Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis, can maintain this function for life, and are the functional units in clinical bone marrow transplantation. The limited HSC numbers not only restricts their clinical use but also several embodiments of experimental research. Recently, a highly defined ex vivo culture system was shown to support several hundredfold expansion of functional in vivo HSC activity. Here, we further explored this system. Over a 3-week culture period, only 0.1% of initially seeded HSCs retained their original phenotype, which contained virtually all functional long-term HSC activity. Despite this low frequency, net HSC expansions were dramatic due to the overall extensive proliferation in cultures. Limited numbers of expanded HSCs allowed for long-term multilineage engraftment of unconditioned hosts, with candidate expanded HSCs rapidly returning to quiescence in this in vivo setting. The ex vivo differentiated progeny from candidate HSCs was rich in progenitor cell activity and allowed for radioprotection from even single cultured HSCs. Finally, clonal barcoding and competitive repopulation experiments demonstrated that successful HSC expansion emanated from rare HSC clones that was only partially predicted by phenotype, demonstrating the large impact of cellular heterogeneity for HSC biology.