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

Abstract

ABSTRACTHematopoietic 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. Limitations in HSC numbers not only restricts their clinical use but also several embodiments of experimental research. A highly defined ex vivo culture system was recently shown to support robust expansion of in vivo HSC activity, prompting us to explore this system further. Over a 3-week culture period, only 0.1% of cultured cells retained the original input HSC phenotype, but these cells contained virtually all functional long-term HSC activity. Despite a low frequency of candidate HSCs, the net functional HSC expansion was large and necessitated several adjustments to the classical competitive repopulation for quantification. The ex vivo differentiated progeny from candidate HSCs was rich in progenitor cell activity and allowed for radioprotection from even single cultured HSCs. Limited numbers of expanded HSCs allowed for long-term multilineage engraftment of unconditioned hosts, with expanded HSCs rapidly returning to quiescence in this in vivo setting. Finally, clonal barcoding and competitive repopulation experiments demonstrated that successful HSC expansion emanated from rare HSC clones that was only partially predicted by phenotype. While a culture system that robustly expands HSC activity have been a long-sought goal in experimental HSC biology, our studies highlight the large impact of cellular heterogeneity also for this aspect of HSC biology.Key pointEx vivo self-renewal is an intrinsic property of rare candidate HSCs, with implications for assessments of HSC activity by transplantation.