Age-associated characteristics of murine hematopoietic stem cells

Abstract

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34(-/low)c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34(-)KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34(-)KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34(-)KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

Figure 1

Limiting dilution analysis based on competitive repopulation. Varying numbers of bone marrow cells from either 2- or 18-mo-old mice were mixed with 2 × 10⁵ bone marrow cells from 2-mo-old mice and injected into lethally irradiated mice. Reconstitution in myeloid and B and T lymphoid lineages was evaluated 12 wk after transplantation. Mice were considered to be negative when percent chimerism for myeloid or B or T lymphoid lineage was <1.0. 1 in 3.2 × 10⁴ cells and 1 in 1.6 × 10⁴ cells was a multilineage repopulating cell for 2-mo-old (▪) and 18-mo-old (•) mice. When uni- and bilineage repopulating cells were included in estimations of frequencies, 1 in 1.0 × 10⁴ cells was a repopulating cell in 18-mo-old mice (○).

Figure 2

Limiting dilution analysis on CD34⁻KSL cells from 18-mo-old mice. Graded numbers of CD34⁻KSL cells were mixed with 2 × 10⁵ bone marrow competitor cells and transplanted into irradiated mice. Peripheral blood cells of the recipients were analyzed for the presence of CD34⁻KSL-derived cells 16 wk after transplantation. The frequencies of multilineage repopulating cells (•) and a total of multilineage repopulating cells and myeloid-dominant repopulating cells (○) were estimated as 1 in 23 and 1 in 13 CD34⁻KSL cells, respectively.

Figure 3

FACS^® profiles for reconstitution analysis. Data are from recipients transplanted with a limited number of CD34⁻KSL cells from 18-mo-old mice. At 16 wk after transplantation, peripheral blood cells were analyzed for contributions of test donor cells, which were distinguished as Ly5.1 cells. Multilineage reconstitution was detected in a mouse transplanted with 20 CD34⁻KSL cells, as shown in A. Myeloid lineage was predominately reconstituted in a mouse transplanted with 10 CD34⁻KSL cells (Table , mouse No. 2), as shown in B. Note good repopulation in all lineages by F1 competitor cells in both cases.

Figure 4

BrdU incorporation by CD34⁻KSL cells. After administration of BrdU to mice, CD34⁻KSL cells (•) and lineage marker–negative cells (○) were isolated from the mice and analyzed for BrdU uptake (A). The data are shown with a function of logarithmic percent of BrdU-negative cells (B).