Defining Hematopoietic Stem and Progenitor Cell Turnover by Analysis of Histone 2B-GFP Dilution

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.