Functional heterogeneity of adult mouse bone marrow hematopoietic stem cells

The mammalian blood-forming system sustains physiologically required levels of mature blood cells by supporting their continuous generation from a rare population of undifferentiated, self-sustaining pluripotent hematopoietic "stem" cells (HSCs). Throughout adult life HSCs are located primarily in the bone marrow. Traditionally, the study of HSCs within larger populations of cells has hampered the direct observation of any unique differentiation or self-renewal properties that might distinguish individual members of the HSC compartment. To circumvent this, I analyzed the number and types of progeny generated from single purified HSCs both in cultures initiated with a single cell and in irradiated mice injected with a single cell. In a first set of experiments of this type, I demonstrated that two growth factor cocktails with the same mitogenic and antiapoptotic activity on HSCs in vitro could have remarkably disparate effects on their concomitant self-renewal behaviour, even within the span of a single cell cycle. In addition, I used high-resolution video monitoring of single purified HSCs cultured in microwell arrays to identify cellular features that were associated with HSC self-renewal in vitro. These parameters include longer cell-cycle times than those of their differentiating progeny and an absence of uropodia on the majority of cells within the clone during the final 12 hours of culture. When combined, these parameters improved by a factor of 2-3-fold the identification of clones found to contain daughter HSCs with longterm in vivo reconstituting ability. Finally, from longitudinal and serial WBC analyses performed on a large number of recipients of single purified HSCs, I found that the adult HSC compartment could be resolved into 4 HSC subtypes, 2 of which stably and autonomously propagate their initial unique patterns of WBC reconstitution through many self-renewal divisions in vivo. I also found that, in vitro, HSCs could rapidly acquire less competitive in vivo reconstitution programs although remarkable symmetry was retained in the reconstitution programs acquired by the daughter HSCs generated in the first 4 days in vitro. These findings provide evidence of intrinsically determined heterogeneity in the differentiation and self-renewal properties of individual HSCs.Medicine, Faculty ofMedical Genetics, Department ofGraduat