Further characterization of CD34-low/negative mouse hematopoietic stem cells.
We have previously reported that in adult mouse bone marrow, CD34low/- 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.
Nakauchi
H
H
Department of Immunology, Institute of Basic Medical Sciences, Ibaraki, Japan. nakauchi@md.tsukuba.ac.jp
Takano
H
H
Ema
H
H
Osawa
M
M
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D002469:Cell Separation; D004268:DNA-Binding Proteins / Q000235:genetics; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation / Q000276:immunology; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D051379:Mice; D011835:Radiation Protection; D018121:Receptors, Cytokine / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics; D014916:Whole-Body Irradiation
D018952:Antigens, CD34; D015415:Biomarkers; D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494712:Gata2 protein, mouse; D018121:Receptors, Cytokine; D014157:Transcription Factors