CDK6 Levels Regulate Quiescence Exit in Human Hematopoietic Stem Cells

Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G0 fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells1, 2 and have been presumed to retain labels such as the nucleotide analogue 5-bromodeoxyuridine (BrdU). However, recently it has been demonstrated that BrdU-retention is neither sensitive nor specific for HSCs3. Here we show that transient, transgenic expression of a Histone2B (H2B)-Green Fluorescent Protein (GFP) fusion protein in mice allows superior labeling of HSCs and permits improved analysis of their turnover in combination with other markers. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a highly stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and suggests that ~ 20% of HSCs turn over at an extremely low rate (≤ 0.8–1.8% per day). Prospective isolation and transplantation of L−K+S+CD48−CD150+ HSCs with different H2B-GFP levels revealed that higher H2B-GFP label retention correlates with superior long-term repopulation potential.

The Thy-1.1loSca-1hiLin-/lo population, representing 0.05% of C57BL/Ka-Thy-1.1 bone marrow, is highly enriched for hematopoietic stem cells and includes all multipotent progenitors in this mouse strain; however, the functional reconstituting activity of this fraction is heterogeneous. Only around 25% of clonal reconstitutions by cells from this population are long term; remaining clones yield transient multilineage reconstitutions. By fractionating based on lineage marker expression, the Thy-1.1loSca-1hiLin-/lo population has been resolved into three subpopulations: Lin-Mac-1-CD4-; Lin-Mac-1loCD4-; and Mac-1loCD4lo. Of these, only the Lin-Mac-1-CD4- population is highly enriched for long-term reconstituting hematopoietic stem cells. A comparison of transient and long-term multipotent progenitors indicates that long-term progenitors have less CFU-S activity, are equally radioprotective, and are less frequently in cell cycle. The ability to predict the longevity of reconstitution based on lineage marker expression indicates that reconstitution potential is deterministic, not stochastic.