Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells.

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34(+)CD38(+) fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

Age-dependent increase in side population distribution within hematopoiesis: implications for our understanding of the mechanism of aging.

It is thought that, as we age, damage to our stem cells may lead to diminished stem cell pool function and, consequently, a reduced organ regeneration potential that contributes to somatic senescence. Stem cells have evolved many antitoxicity mechanisms, and certain mechanisms may be utilized to isolate hematopoietic stem cells. One method exploits the activity of the ATP-binding cassette/G2 transporter to efflux Hoechst 33,342 and results in a stem cell population known as the side population (SP). The SP subset represents a remarkable enrichment for hematopoietic stem cells and provides an opportunity to re-evaluate age-based changes in hematopoietic stem cells. We report here that the frequency of SP cells steadily increases with age, as does the proportion of Lin(-)/Sca-1(+)/c-kit(+) cells that is capable of Hoechst efflux. Phenotyping, progenitor, and long-term repopulation assays have indicated that SP cells in older mice are still stem cells, albeit with a lower homing efficiency than SP cells from younger mice. Analysis of apoptosis within SP cells has revealed an apoptosis-resistant population in SP cells from old mice. Gene expression analysis has determined that SP cells from old mice have a reduced expression of apoptosis-promoting genes than SP cells from young mice. This increase in SP cells with age seems to be an intrinsic property that may be independent of the age of the microenvironment (niche), and our data might provide some clues as to how this alteration in the proportion of stem/progenitor cells occurs. A possible selection-based mechanism of stem cell pool aging is discussed.

AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) assay is the current model for assessment of human normal and leukemic stem cells. We explored why 51% of 59 acute myeloid leukemia (AML) patients were unable to initiate leukemia in NOD/SCID mice. Increasing the cell dose, using more permissive recipients, and alternative tissue sources did not cause AML engraftment in most previously nonengrafting AML samples. Homing of AML cells to the marrow was the same between engrafters and nonengrafters. FLT3 internal tandem duplication (ITD) and nucleophosmin mutations occurred at a similar frequency in engrafters and nonengrafters. The only variable that was related to engraftment ability was the karyotypically defined risk stratification of individual AML cases. Of interest, follow-up of younger patients with intermediate-risk AML revealed a significant difference in overall survival between NOD/SCID engrafting and nonengrafting AMLs. Hence, the ability of AML to engraft in the NOD/SCID assay seems to be an inherent property of AML cells, independent of homing, conditioning, or cell frequency/source, which is directly related to prognosis. Our results suggest an important difference between leukemic initiating cells between engrafting and nonengrafting AML cases that correlates with treatment response.

Multiparameter analysis of murine bone marrow side population cells.

We describe the multiparameter flow cytometric analysis of the relationship between side population (SP) formation and well-characterized, antigen-defined stem cell subsets. We also compared the competitive repopulation ability of subsets defined by the SP profile to those identified by antigenic markers. The vast majority of SP cells possessed a primitive cell phenotype (c-kit+, SCA-1+, Thy-1+, CD31+, CD135neg, lineage neg), but only a minority of antigen-defined subsets were SP cells. Hence, although SP cells are identified independently of antigenic markers, they are not distinct from established stem cell phenotypes, but are a small subset of them. Approximately half of SP cells expressed CD34 at readily detectable levels, and one-third of SP cells possessed the primitive c-kit+, SCA-1+, lineage neg, CD34neg cell phenotype. Since most SP cells are a subset of c-kit+, Thy-1+, lineage neg, SCA-1+ cells (KTLS), we determined whether the SP cell subset represents a further enrichment in long-term repopulating cell content. The SP+ subset of KTLS+ cells was more enriched for competitive repopulation units than the SP- fraction of KTLS+ cells. Hence, the SP cell fraction highlights a subset of the long-term repopulating cells found within the already highly purified KTLS fraction.