Heterogeneity of leukemia stem cell candidates at diagnosis of acute myeloid leukemia and their clinical significance.

Leukemia stem cell candidates (LSCC) can be enriched from patients with acute myeloid leukemia by high aldehyde dehydrogenase (ALDH) activity and CD34 expression. We have previously demonstrated the leukemia-initiating activity of ALDH(bright) cells in xenograft transplantation models, as well as in vitro. Applying single-cell long-term culture-initiating cell assays, we have correlated the functional properties of individual cells within this LSCC population and the respective phenotypes. To define their biologic significance, we also analyzed the relationship between LSCC at diagnosis to long-term clinical outcomes. The median percentage of ALDH(bright) cells among 101 acute myeloid leukemia patients was 0.51% (range, 0.01-12.90%). Single-cell long-term culture-initiating cell assays, followed by genetic analysis of the progeny cells, showed that the leukemia-initiating activity was found in the ALDH(bright)/CD34(high) subset and, to a lesser extent, in ALDH(bright)/CD34(low) or ALDH(bright)/CD34(-) subsets. Nevertheless, the frequency of ALDH(bright) cells at diagnosis correlated significantly with the persistence of leukemia after induction chemotherapy (n = 84, Spearman R = 0.3261; p < 0.0025). In the multivariate model, frequency of ALDH(bright) cells was the strongest prognostic marker (p = 0.0095) affecting overall survival (hazard ratio = 9.107). LSCC are heterogeneous and best reflected by ALDH activity. The frequency of ALDH(bright) cells at diagnosis is a significant prognostic marker for acute myeloid leukemia.

Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan.

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm(2) is exceeded. At shear stress ∼1 dyn/cm(2), the cell rolling speed reaches a maximum of 15 µm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.

Innovative method for quantification of cell-cell adhesion in 96-well plates.

Cell adhesion is an important part of many complex biological processes. It plays crucial roles in cancer, development, and maintenance of stem cell compartment. The measurement of adhesion under experimental conditions might provide important information for cell biology. There are several protocols to measure adhesion, usually based on washing or shaking to remove non-adherent cells. Here, we describe a quantification method based on gravitational force to measure adhesion in a 96-well format. Non-adherent cells are separated and only vital cells are quantified with a colorimetric assay. As example we provide the quantification of cell-cell interaction with blocking function antibodies for CD44, an N-cadherin antagonists and the stromal cell derived factor-1 alpha (SDF-1). This method facilitates fast and reliable measurement of cell adhesion in multiwell format for screening assays.

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor.

BACKGROUND AIMS: Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS: We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS: The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P < 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally, two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS: A combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets.

Differential survival of AML subpopulations in NOD/SCID mice.

OBJECTIVE: Leukemia-initiating cells can retrospectively be defined by tumorigenicity in immunodeficient mice and be characterized by surface markers. The latter still being discussed for acute myeloid leukemia (AML), nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to evaluate long-time reconstitution and expansion of AML subpopulations. MATERIALS AND METHODS: Bone marrow cells from patients with AML were separated according to CD34 expression, aldehyde dehydrogenase (ALDH) activity, and divisional kinetics in comparison to cord blood-derived CD34(+) hematopoietic stem cells, evaluating survival and expansion in NOD/SCID mice. The AML long-term surviving capacity of subpopulations recovered from NOD/SCID mice was confirmed by ex vivo survival. RESULTS: AML mononuclear cells were detected in bone marrow and spleen of NOD/SCID mice 12 weeks after transplantation. The majority of recovered cells were CD34(+) and significantly more CD34(+) cells were recovered after application of ALDH(bright) (high ALDH activity), CD34(+), or slowly dividing (PKH(bright)) than after ALDH(dim), CD34(-), or fast dividing (PKH(dim)) cell application. CD123(+), CD63(+), and CD44v7(+) cells were also more abundant after the transfer of ALDH(bright) or CD34(+) AML mononuclear cells. In the spleen, large AML cell clusters were only recovered after ALDH(bright), CD34(+), or PKH(bright) cell transfer. Importantly, in secondary long-term in vitro cultures, quite exclusively CD34(+) AML mononuclear cells survived and expanded. CONCLUSIONS: Separation of ALDH(bright), CD34(+), or PKH(bright) cells enriches for AML long-term surviving capacity, which reside in the CD34(+) subpopulation, as rather exclusively CD34(+) cells survived and expanded in vivo and ex vivo. Long-term survival capacity may be supported by CD44v7 expression.

Improved T and B cell recovery by the transfer of slowly dividing human hematopoietic stem cells.

Human hematopoietic stem cells giving rise to long term initiating cells in vitro are enriched in a CD34(+) slow dividing fraction (SDF). Here, we tested reconstitution and multilineage differentiation of this CD34(+) SDF in NOD/SCID mice. In the bone marrow a slightly higher percentage of human hematopoietic progenitors were recovered after the transfer of the SDF compared to the fast dividing fraction. Instead, T cell maturation in the rudimentary thymus and lymph node repopulation was only initiated by the SDF. The capacity of the SDF to differentiate and mature in the patients' thymus could provide an advantage in immunocompetence recovery.

Aldehyde dehydrogenase activity among primary leukemia cells is associated with stem cell features and correlates with adverse clinical outcomes.

OBJECTIVE: Animal models have provided evidence for the existence of leukemia stem cells (LSC). However, prospective isolation of human LSC from patients with acute myeloid leukemia (AML), as well as the assessment of their clinical significance, has remained a major challenge. MATERIALS AND METHODS: We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)), which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS: The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients, the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; p<0.01), a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; p<0.05), higher numbers of long-term culture-initiating cell colonies in vitro (n=25; p<0.01), and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; p<0.05). Above all, we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (p<0.05). CONCLUSION: A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome.

Differential expression of cancer-related genes by single and permanent exposure to bone morphogenetic protein 2.

PURPOSE: Bone morphogenetic proteins (BMPs) are multifunctional regulators of various cell functions. The BMP-signalling network plays a pivotal role during embryogenesis and tumorigenesis. BMPs, e.g. BMP-2 exert their biological function in a time and concentration-dependent manner but also modulated by the context of the cellular microenvironment. In this study, we investigated the effect of a steady high level of BMP-2 versus a single application of BMP-2 on the breast cancer cell line MCF-7. METHODS: The effect of the incubation regimes was analysed by DNA microarray expression profiling. Data were verified by real-time PCR. The protein expression of apoptosis-related genes was studied by western blot analysis. RESULTS: We found a clear difference in the altered gene expression between the constant high level and the single application of BMP-2. After grouping the genes of interest into the biological processes of Gene Ontology, the group of apoptosis-related genes like BAX, BAG5 or PKR, was predominantly affected under the single-application regime of BMP-2. Among these protein kinase R was the most prominently regulated. Further studies on the protein level showed activation of PKR after 4 h with a subsequent enhanced phosphorylation of the PKR substrate eIF2alpha for several hours. CONCLUSIONS: The duration of treatment and the concentration of BMP-2 affect the global expression pattern of MCF-7 cells. Among the regulated cancer-related genes, the cohort of the apoptosis-related genes showed the pronounced alterations. Our data point to a novel role of BMP-2 in the regulation of the PKR pathway in tumorigenesis.