Figure 2.

Old and young BM cells with HSC markers have similar clonogenic ability in liquid culture but have reduced activity in stromal co-cultures. (A) 60–120 purified Lin^loSca1⁺cKit⁺CD48⁻CD34⁻EPCR⁺CD150⁺ (LSK48⁻34⁻E⁺150⁺) and LSK48⁻150⁺ cells from each of five young or six old mice were seeded individually as single cells into liquid cultures supplemented with stem cell factor and IL-11. Shown is the mean proportion of single purified cells from the individually tested young and old mice that generated clones of at least 5,000 cells within 2 wk of culture. (B) 60–120 purified LSK48⁻34⁻E⁺150⁺, LSK48⁻150⁺, and LSK48⁺150⁻ cells from young or old mice were seeded as single cells onto FBMD stroma and scored weekly for the presence or absence of cobblestone areas. Shown is the mean proportion of single purified cells from individually tested young (n = 7–9) and old (n = 12–20) mice that generated cobblestone areas at day 7 or later (CAFCd7+). (C) Dots represent individual young (n = 9) or old (n = 19) mice and show the size of the LSK48⁻34⁻E⁺150⁺ pool (as described in Fig. 1) and the clonogenic efficiency in stromal co-culture (as shown in B). (D) LSK48⁻34⁻E⁺150⁺ (HSC containing), LSK48⁻34⁺150⁻ (more primitive MPPs), and LSK48⁺34⁺150⁻ (less primitive MPPs) were isolated from young BM and seeded individually onto FBMD stroma and scored weekly for the presence or absence of cobblestone areas, which are characteristic of proliferating primitive cells. Data represent a minimum of 500 wells per cell type from a total of 10 individual young mice. (E) 60 purified LSK48⁻34⁻E⁺150⁺ cells from each of 9 young and 19 old mice were seeded individually as single cells onto FBMD stroma and scored weekly for 11–13 wk for the presence or absence of cobblestone areas. Shown is the mean proportion of single purified LSK48⁻34⁻E⁺150⁺ cells from the individually tested young and old mice that generated cobblestone areas at any time point day 35 or later (CAFCd35+). (F) Proportion of LSK48⁻34⁻E⁺150⁺ cells with CAFCd35+ activity (described in E) that first generated cobblestone areas at the specified time after seeding. P < 0.001 for old (n = 187) versus young (n = 149) CAFCd35+, Mann-Whitney U test. Error bars on all panels represent 95% confidence intervals of the mean. ***, P < 0.001, unpaired two-tailed Student’s t test.