Objective-To test function of hematopoietic stem cells (HSCs) in vivo in C57BL/6 (B6) and Trp53-deficient (Trp53 null) mice by using two HSC enrichment schemes.Methods-Bone marrow (BM) Lin -CD41 -CD48 -CD150 + (SLAM, signaling lymphocyte activation molecules), Lin -CD41 -CD48 -CD150 -(SLAM -) and Lin -Sca1 + CD117 + (LSK) cells were defined by fluorescence activated cell staining (FACS). Cellular reactive oxygen species (ROS) level was also analyzed by FACS. Sorted SLAM, SLAM -and LSK cells were tested in vivo in the competitive repopulation (CR) and serial transplantation assays.Results-The SLAM cell fraction was 0.0078 ± 0.0010% and 0.0135 ± 0.0010% of total BM cells in B6 and Trp53 null mice, and was highly correlated (R 2 = 0.7116) with LSK cells. CD150 + BM cells also contained more ROS low cells than did CD150 -cells. B6 SLAM cells repopulated recipients much better than B6 SLAM-cells, showing high HSC enrichment. B6 SLAM cells also engrafted recipients better than Trp53 null SLAM cells in the CR and the follow-up serial transplantation assays. Similarly, LSK cells from B6 donors also had higher repopulating ability than those from Trp53 null donors. However, whole BM cells from the same B6 and Trp53 null donors showed the opposite functional trend in recipient engraftment. Hematopoietic stem cells (HSCs) reside in fetal liver, cord blood and adult bone marrow (BM) in very low frequencies but perform a vital function of sustaining life-long production of all mature blood cells [1][2][3][4][5]. Various methods have been developed to enrich HSCs for functional studies as well as for clinical transplantation [6][7][8][9][10]. HSCs lack the expression of cell surface molecules for lineage-specific mature cells, and are therefore Lin - [6,7,10]. In mouse models, HSCs usually express stem cell antigen 1 (Sca1 + ) and c-Kit (Kit + , CD117), a trans-membrane tyrosine kinase that serves as a receptor for stem cell factor [10][11][12]. These findings led to the establishment of Lin -Sca1 + Kit + (LSK) as a standard marker set for HSCs, although other Conclusion-Both

Human bone marrow (BM) failure mediated by the immune system can be modeled in mice. In the present study, infusion of lymph node (LN) cells from C57BL/6 mice into C.B10-H2b/LilMcd (C.B10) recipients that are mismatched at multiple minor histocompatibility Ags, including the immunodominant Ag H60, produced fatal aplastic anemia. Declining blood counts correlated with marked expansion and activation of CD8 T cells specific for the immunodominant minor histocompatibility Ag H60. Infusion of LN cells from H60-matched donors did not produce BM failure in C.B10 mice, whereas isolated H60-specific CTL were cytotoxic for normal C.B10 BM cells in vitro. Treatment with the immunosuppressive drug cyclosporine abolished H60-specific T cell expansion and rescued animals from fatal pancytopenia. The development of BM failure was associated with a significant increase in activated CD4+CD25+ T cells that did not express intracellular FoxP3, whereas inclusion of normal CD4+CD25+ regulatory T cells in combination with C57BL/6 LN cells aborted H60-specific T cell expansion and prevented BM destruction. Thus, a single minor histocompatibility Ag H60 mismatch can trigger an immune response leading to massive BM destruction. Immunosuppressive drug treatment or enhancement of regulatory T cell function abrogated this pathophysiology and protected animals from the development of BM failure.

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Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin(-)Kit(+)CD34(-) parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS(-) cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.