Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells

“…to Gr-1+SSChi cells at 24 wk after transplant were considered to be repopulated with HSCs and were counted as positive for the purposes of the limiting dilution calculation, performed using the Web-based calculator provided at http://bioinf.wehi.edu .au/software/elda/ (Hu and Smyth, 2009).”

“To confirm the utility of this measurement, we sorted three LSK subpopulations from young BM: LSK4834E+150+, LSK4834+150, and LSK48+34+150, which are known to contain HSCs, more primitive multipotent progenitors (MPPs), and less primitive MPPs, respectively (Wilson et al., 2008).”

“…of these differences is that upon transplantation; old HSCs tend to have a myeloid-skewed blood cell production caused by a decreased ability to produce lymphoid cells (Sudo et al., 2000; Kim et al., 2003; Liang et al., 2005; Rossi et al., 2005; Cho et al., 2008; Beerman et al., 2010a).”

“Consistent with earlier studies (Morrison et al., 1996; Sudo et al., 2000; Rossi et al., 2007b), the median proportion of LinloSca1+cKit+ (LSK) cells within the BM increased 1.9-fold in old mice (Fig.”

“Exciting recent developments in purification and assays for human HSCs (Notta et al., 2011) finally open up the possibility for similar interrogation of the aged human HSC pool at a clonal level.”

“As a strict measure for long-term, active hematopoiesis, we considered a mouse to be transplanted with an HSC only if there was a minimum 1% donor contribution to the short-lived (Pillay et al., 2010) granulocyte population (defined as Gr-1+SSChi) at 24 wk after transplant.”

“…expressing HSC-associated markers is contaminated with primitive progenitors that are detected by in vitro assays but lack repopulating ability in vivo (Sudo et al., 2000) or that HSCs from old mice have a homing and/or engraftment defect and therefore remain undetected (Morrison et al., 1996).”

“…expressing HSC-associated markers is contaminated with primitive progenitors that are detected by in vitro assays but lack repopulating ability in vivo (Sudo et al., 2000) or that HSCs from old mice have a homing and/or engraftment defect and therefore remain undetected (Morrison et al., 1996).”

“This decision was taken to ensure that any cells with robust self-renewal potential but lacking appreciable lymphoid differentiation potential, as have been reported to exist in both young (Dykstra et al., 2007; Morita et al., 2010) and old mice (Sudo et al., 2000), would not be excluded.”

“Specifically, myeloid-dominant HSCs are thought to accumulate in the old HSC pool at the expense of lymphoid-dominant and lineage-balanced HSCs (Cho et al., 2008; Muller-Sieburg and Sieburg, 2008; Beerman et al., 2010a; Challen et al., 2010).”

“Support for the latter is provided by a study of short-term HSC homing that reported a twofold lower ability of functionally defined HSCs from old mice to home to the BM or spleen within a 24 h period (Liang et al., 2005).”

“Specifically, myeloid-dominant HSCs are thought to accumulate in the old HSC pool at the expense of lymphoid-dominant and lineage-balanced HSCs (Cho et al., 2008; Muller-Sieburg and Sieburg, 2008; Beerman et al., 2010a; Challen et al., 2010).”

“This is a more stringent definition than is normally applied and would be expected to exclude virtually all clones in which robust self-renewal had not occurred (Dykstra et al., 2007; Kent et al., 2009).”

“The relative contributions of each of these factors continue to be important areas of research and debate (Liu and Rando, 2011).”

“…the functional properties of individual HSCs gradually decline as the result of an accumulation of cellular damage such as the accrual of DNA lesions (Rossi et al., 2007a; Mohrin et al., 2010; Yahata et al., 2011) or epigenetic dysregulation (Bennett-Baker et al., 2003; Chambers et al., 2007).”

“Recently, Takizawa et al. (2011) reported that HSCs with extensive proliferative history were particularly prone to return to a quiescent state.”

“However, the identification of separate populations of intrinsically lineage-biased HSCs (Müller-Sieburg et al., 2002; Dykstra et al., 2007) has suggested a different model in which the clonal composition of the HSC pool, rather than individual HSCs, changes with age.”

“The former hypothesis is supported by observations of improved functional frequencies when alternate marker combinations are used to purify old HSCs (Yilmaz et al., 2006).”

“To do this, we purified LSK48150+ cells from old and young mice transgenic for CFP, GFP, or dsRed, combined them at a known ratio, and injected them into 2696 Functional defects of aged murine HSCs | Dykstra et al. JEM Vol. 208, No. 13 Article 2697 that excluded CD150 cells (Kent et al., 2009; Beerman et al., 2010a; M…”

“…into 2696 Functional defects of aged murine HSCs | Dykstra et al. JEM Vol. 208, No. 13 Article 2697 that excluded CD150 cells (Kent et al., 2009; Beerman et al., 2010a; Morita et al., 2010) and a strict requirement for donor-derived granulocyte production at 24 wk after transplant to score mice…”

“Transplants of old BM were shown to outperform the same number of cells from young mice (Harrison, 1983), and this was later determined to be caused by an age-related increase in the concentration of HSCs in the BM (Harrison et al., 1989).”

“…of these differences is that upon transplantation; old HSCs tend to have a myeloid-skewed blood cell production caused by a decreased ability to produce lymphoid cells (Sudo et al., 2000; Kim et al., 2003; Liang et al., 2005; Rossi et al., 2005; Cho et al., 2008; Beerman et al., 2010a).”

“Consistent with earlier studies (Morrison et al., 1996; Sudo et al., 2000; Rossi et al., 2007b), the median proportion of LinloSca1+cKit+ (LSK) cells within the BM increased 1.9-fold in old mice (Fig.”

“Transplants of old BM were shown to outperform the same number of cells from young mice (Harrison, 1983), and this was later determined to be caused by an age-related increase in the concentration of HSCs in the BM (Harrison et al., 1989).”

“Vol. 208 No. 13 2691-2703 www.jem.org/cgi/doi/10.1084/jem.20111490 2691 Organismal aging is accompanied by a general decline in many tissues and organs.”

“This finding challenges the notion that the myeloid-dominant HSCs that accumulate with age are functionally equivalent to those in young mice (Cho et al., 2008; Muller-Sieburg and Sieburg, 2008).”

“…et al. 2000; Rossi et al. 2005) that shows decreased regenerative potential (Morrison et al. 1996; Sudo et al. 2000; Kim et al. 2003; Rossi et al. 2005; Dykstra et al. 2011) and a myeloid-skewed differentiation potential (Sudo et al. 2000; Kim et al. 2003; Liang et al. 2005; Rossi et al. 2005).”

“Despite the increase in their number, myeloid-biased stem cells are not as robust as their young counterparts (24), which in turn could underlie the numerous age-related deficiencies observed in mature myeloid cells such as neutrophils and macrophages (25, 26).”