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

Abstract

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.

Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion

“…Cell cycle analysis was performed to address whether an increase in cell cycle rate might represent a selective force capable of propelling Slamf1 high HSCs to predominate the stem over time. Consistent with studies detailing the deeply quiescent nature of adult HSCs (29)(30)(31), both HSC subsets were predominantly out of cycle, in contrast to downstream progenitors, which were rapidly cycling (Fig. 3E).…”

Cell cycle regulation in hematopoietic stem cells

“…In vivo experiments assessing cell cycle activity (by measuring retention of BrdU or histone 2B (H2B)-GFP expression pulses) in mature HSCs suggest a notable heterogeneity in the degree to which HSCs are quiescent Foudi et al, 2009). These studies propose the subfractionation of the HSC compartment into "dormant" and "activated" phenotypes with distinct rates of cell cycle entry, comprising 5-10% and 90-95% of the HSC pool, respectively (Fig.…”

“…p21 Cip is expressed at somewhat greater levels in adult HSCs relative to their differentiated progeny or to fetal HSCs (Passegué et al, 2005;Bowie et al, 2007). Although a role for p21 Cip in regulating HSC quiescence had initially been suggested (Cheng et al, 2000a), more recent reports using different mouse backgrounds and/or methodologies suggest that the function of p21 Cip in regulating HSC cell cycle activity may be restricted to periods of stress rather than during homeostasis (Cheng et al, 2000a;van Os et al, 2007;Foudi et al, 2009). Aside from p21 Cip , analysis of p27 Kip1 -deficient mice suggests that p27 Kip1 deficiency alone has a limited impact on HSC function and instead appears to affect the cell cycle activity of more committed progenitor populations (Cheng et al, 2000b).…”

Prospective isolation of resident adult human mesenchymal stem cell population from multiple organs

“…The most popular cell source is bone marrow (BM) stem cells, which consist of hematopoietic stem cells (HSCs) and mesenchymal stem/ stromal cells (MSCs). HSCs are present in the BM niche in a state of quiescence [1][2][3]. MSCs reside in the non-hematopoietic fraction and can be cultured to form fibroblast-like colonies (colony-forming unit fibroblasts: CFU-Fs) in vitro [4][5][6][7].…”