Further characterization of CD34-low/negative mouse hematopoietic stem cells

We have previously reported that in adult mouse bone marrow, CD34(low/-) c-kit(+) Sca-1(+) lineage markers negative (Lin(-)) (CD34(-)KSL) cells represent hematopoietic stem cells with long-term marrow repopulating ability whereas CD34(+) c-kit+ Sca-1(+) Lin(-) (CD34(+)KSL) cells are progenitors with short-term reconstitution capacity. To further characterize cells in those two populations, relative expression of various genes were examined by reverse transcriptase polymerase chain reaction (RT-PCR), In CD34(-)KSL cells, none of the genes studied was found to be expressed with the exception of GATA-2, IL-1R alpha, IL-2R gamma, AIC-2B, c-kit, EPO-R, and c-mpl, In contrast, expression of GATA-1 and all cytokine receptor genes examined except IL-2R beta, IL-7R alpha and IL-9R alpha were found in CD34(+)KSL. The difference between these two populations was also shown in single cell culture analysis of these cells. When cells were clone-sorted and cultured in the presence of SCF, IL-3 and EPO, CD34(-)KSL cells required much more time to undergo the first cell division than CD34(+)KSL cells. Dormancy and random fashion of cell division by CD34(-)KSL cells were also evident by the analysis of the second cell division, which was found to be delayed and unsynchronous compared with CD34(+)KSL cells. Clonal culture analysis showed that CD34(-)KSL cells were more potent in proliferation and multilineage differentiation capacities than CD34(+)KSL cells. In a paired-daughter cell experiment, 75% of CD34(-)KSL and 50% of CD34(+)KSL paired-daughter-derived colonies were nonidentical with wide variety of lineage combinations. Taken together, these data support our previous notion that CD34(-)KSL cells are at higher rank in hematopoietic hierarchy than CD34(+)KSL cells, In addition, our results using highly enriched stem cell population directly obtained from mouse bone marrow support the proposed stochastic nature of lineage commitment.