Hematopoietic Stem Cell Cycling Dynamics In Steady State and Upon Hematopoietic Challenge

Abstract 572

Life-long blood production is maintained by a small fraction of hematopoietic stem cells (HSCs). Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assays with 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic mouse models (Wilson et. al., 2008; Foudi et. al., 2009). While the former studies showed that all HSCs equally divide and likely contribute to blood formation (clonal maintenance), the latter suggested that some HSCs divide frequently and contribute to blood formation until cell death or full differentiation, while some HSCs are quiescent and then get activated to follow the same fate as frequently dividing ones (clonal succession). However, due to low resolution, none of the labeling techniques used were able to track single cell divisions. Furthermore, methods used might have direct influence on cycling activity of HSCs. Thus it remains to be determined a) if HSC divide continuously, sequentially or repetitively and contribute to steady-state hematopoiesis, b) what is a relationship between divisional history and repopulating ability, and c) how self-renewal and differentiation capacity of HSC is impacted by naturally-occurring severe hematopoietic challenges as infections. To address this directly, we set up a high resolution non-invasive in vivo HSC divisional tracking assay with CFSE (carboxyfluorescein diacetate succinimidyl ester). We here show that i.v. transfer of CFSE-labeled HSCs into non-conditioned congenic recipient mice allows evaluation of steady-state HSC cycling-dynamics as CFSE is equally distributed to daughter cells upon cellular division. Transfer of Lin-c-kit+Sca-1+ cells (LKS) into non-irradiated mice revealed non- and 1–7x divided LKS in recipient bone marrow over 20 weeks. To test in vivo limiting dilution and single cell HSC potential, non- or ≥5x divided cells were sorted based on divisional history from primary recipients at different weeks after transplantation, and transplanted into lethally irradiated secondary recipients. Single non-divided LKS at 3 weeks post primary transfer was able to multi-lineage repopulate 24% of recipients long-term, while 50 of ≥5x divided LKS did not engraft. Interestingly, both non- and ≥5x divided LKS at 7 or 12–14 weeks after primary transfer engrafted and showed fluctuating contribution to multi-lineage hematopoiesis over serial transplantation. Mathematical modeling based on limiting dilution transplantation, revealed no evidence for a dichotomy of biologically defined HSCs in different groups. Instead, steady-state serial transplantation with temporary fast-cycling cells revealed that they can slow down over time, suggesting dynamically changing cycling activity of HSC. We next tested the effects of hemato-immunological challenge on HSC proliferation. Mice transplanted with CFSE-labeled LKS cells were repetitively treated with LPS. Analysis 8 days after final LPS injection, i.e. three weeks after steady-state transplantation revealed that all LKS entered cell cycle and the number of ≥5x divided LKS was increased. Secondary transplantation showed that 2–4 time and ≥5x divided LKS from LPS-treated mice reconstituted multi-lineage hematopoiesis whereas both fractions from control mice failed to engraft. This data clearly indicate that HSCs are activated from quiescence upon LPS challenge and provide evidence, that naturally-occurring hemato-immunological challenges, such as gram-negative bacterial infection induces proliferation and self-renewal of HSCs. Our data suggest in contrast to previously proposed concepts, a novel “dynamic repetition” model for HSC cycling activity and blood formation where some HSCs participate in hematopoiesis for a while, subsequently enter a resting phase and get reactivated again to contribute to blood formation in repetitive cycles, leading to homogenous total divisional history of all HSCs at end of life. These findings might represent a biological principle that could hold true for other somatic stem cell-sustained organ-systems and might have developed during evolution to ensure equal distribution of work-load, efficient recruitment of stem cells during demand, and reduction of risk to acquire genetic alterations or fatal damage to the whole HSC population at any given time.