We have purified hematopoietic stem cells (HSCs) from the bone marrow of old mice and compared their properties to HSCs in young and middle-aged mice. Single, reconstituting HSCs (by limit dilution) from old and young mice exhibited indistinguishable progenitor activities in vivo. HSCs were five times as frequent in the bone marrow of old mice; however, HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients. HSCs in young and middle-aged mice rarely were in the S/G2/M phases of the cell cycle, but HSCs in old mice were frequently in cycle. We speculate that the unexpected proliferation of HSCs in old mice might be related to the increased incidence of leukemia in old mice. HSCs change with age, but it is unknown whether these changes are determined intrinsically or caused by the aging of their environment.

Thy-1°oSca-l+Lin-Mac-l+CD4-cells have been isolated from the livers of C57BL-Thy-1.1 fetuses. This population appears to be an essentially pure population of hematopoietic stem cells (HSC), in that injection of only six cells into lethally irradiated adult recipients yields a limit dilution frequency of donor cell-reconstituted mice. Sixtyseven to 77% of clones in this population exhibit long-term multilineage progenitor activity. This population appears to include all long-term multilineage reconstituting progenitors in the fetal liver. A high proportion of cells are in cycle, and the absolute number of cells in this population doubles daily in the fetal liver until 14.5 days postcoitum. At 15.5 days postcoitum, the frequency of this population falls dramatically. Long-term The developmental and self-renewal potentials of mouse hematopoietic progenitors can be tested at a clonal level in vivo (1). Long-term self-renewing and transiently self-renewing multipotent progenitors can be independently purified from adult mouse bone marrow based on differences in cell surface marker expression (2-4). These populations include all multipotent progenitors in the strain of mouse studied (5) and appear to form a lineage from the earliest isolatable stem cell to the most differentiated multipotent progenitor prior to lineage commitment (unpublished data).The mammalian fetal liver contains hematopoietic stem cells (HSC) capable of long-term multilineage reconstitution of adults (6, 7). The existence of fetal liver progenitors capable of long-term self-renewal and clonal multilineage reconstitution was unambiguously demonstrated in retroviral marking experiments in mice (8-10). Thy-1lOLin-/lOSca-1± cells, representing -0.05% of mouse fetal liver, are highly enriched for multipotent progenitors (refs. 11 and 12; K. Ikuta, personal communication). Long-term reconstituting multipotent progenitors have also been greatly enriched from mouse fetal liver by isolating AA4.1+Linlo (8) or AA4.1+LinloSca-1+ cells (13). AA4.1+LinloSca-1+ cells represent only 0.05-0.08% of fetal liver cells, but the relative representations of long-term reconstituting clones and other progenitors within this population were not reported (13).We set out to purify mouse fetal liver HSC to determine whether multipotent progenitors segregated into populations phenotypically and functionally similar to those described in mouse bone marrow. MATERIALS AND METHODSMouse Strains. The C57BL/Ka-Thyl.1 (Ly5.2 and Ly5.1 strains) and C57BL/J-Ly5.1 (Thyl.2) mouse strains were bred and maintained at the animal care facility at the Stanford School of Medicine. All mice were maintained on acidified water (pH 2.5). Mice used as bone marrow donors were 6-12 weeks old, and irradiated recipient mice were more than 8 weeks old. \ Antibodies. The antibodies used in immunofluorescence staining included 19XE5 (anti-Thyl.1), AL1-4A2 (anti-Ly5.2), 2B8 (anti-c-kit), and E13 (anti-Sca-1) (14). For purposes of this paper, lineage marker antibodies include KT31.1 (anti-CD3), 5...

All multipotent hematopoietic progenitors in C57BL-Thy-1.1 bone marrow are divided among three subpopulations of Thy-1.1(lo) Sca-1+ Lin(-/lo) c-kit+ cells: long-term reconstituting Mac-1- CD4- c-kit+ cells and transiently reconstituting Mac-1(lo) CD4- or Mac-1(lo) CD4(lo) cells. This study shows that the same populations, with similar functional activities, exist in mice whose hematopoietic systems were reconstituted by hematopoietic stem cells after lethal irradiation. We demonstrate that these populations form a lineage of multipotent progenitors from long-term self-renewing stem cells to the most mature multipotent progenitor population. In reconstituted mice, Mac-1- CD4- c-kit+ cells gave rise to Mac-1(lo) CD4- cells, which gave rise to Mac-1(lo) CD4(lo) cells. Mac-1- CD4- c-kit+ cells had long-term self-renewal potential, with each cell being capable of giving rise to more than 10(4) functionally similar Mac-1- CD4- c-kit+ cells. At least half of Mac-1(lo) CD4- cells had transient self-renewal potential, detected in the spleen 7 days after reconstitution. Mac-1(lo) CD4(lo) cells did not have detectable self-renewal potential. The identification of a lineage of multipotent progenitors provides an important tool for identifying genes that regulate self-renewal and lineage commitment.