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).
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We thank Laura Prickett and Kat Folz-Donahue from the HSCI Flow-core at Massachusetts General Hospital (MGH) for their expert assistance, Geoffrey Wahl for providing the H2B-GFP cDNA, and Matthias Stadtfeld, Wenjun Guo and Ben Wittner for valuable input. We thank Linheng Li for the kind gift of biotinylated MNCD-2. We thank Mark Kiel and Sean Morrison for sharing unpublished results and for the gift of excised bone marrow of conditional N-cadherin mutant mice. We thank Glenn Radice for permission to use bone marrow from conditional N-cadherin mutant mice. This work was supported by a contribution from the Ellison Foundation to MGH startup funds for H.H., a seed grant by Harvard Stem Cell Institute to H.H. and by RO1CA122726. H.H. is the recipient of an ASH Scholar Award. K.H. was supported by the NIH Director's Innovator Award, the Harvard Stem Cell Institute, the Kimmel Foundation and the V Foundation. A.F. was the recipient of an MGH ECOR Fund for Medical Discovery Award.
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Foudi, A., Hochedlinger, K., Van Buren, D. et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27, 84–90 (2009). https://doi.org/10.1038/nbt.1517
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