Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells

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Abstract
Foudi et al. report a method for monitoring the turnover of hematopoietic stem cells that has several advantages over BrdU labeling. Using drug-inducible expression of a histone 2B–GFP fusion protein, which permits a more sensitive analysis of division history, the authors detect hematopoietic stem cells that cycle at a very slow rate. 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 thymidine analog 5-bromodeoxyuridine (BrdU). However, BrdU retention is neither a sensitive nor specific marker for HSCs3. Here we show that transient, transgenic expression of a histone 2B (H2B)–green fluorescent protein (GFP) fusion protein in mice has several advantages for label-retention studies over BrdU, including rapid induction of H2B-GFP in virtually all HSCs, higher labeling intensity and the ability to prospectively study label-retaining cells, which together permit a more precise analysis of division history. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and showed that ∼20% of HSCs divide at an extremely low rate (≤0.8–1.8% per day).