Notch signalling is critical for haemopoietic stem cell (HSC) self-renewal and survival. The role of Notch signalling has been reported recently in chronic myeloid leukaemia (CML) - a stem cell disease characterized by BCR-ABL tyrosine kinase activation. Therefore, we studied the relationship between BCR-ABL and Notch signalling and assessed the expression patterns of Notch and its downstream target Hes1 in CD34+ stem and progenitor cells from chronic-phase CML patients and bone marrow (BM) from normal subjects (NBM). We found significant upregulation (p<0.05) of Notch1, Notch2 and Hes1 on the most primitive CD34+Thy+ subset of CML CD34+ cells suggesting that active Notch signalling in CML primitive progenitors. In addition, Notch1 was also expressed in distinct lymphoid and myeloid progenitors within the CD34+ population of primary CML cells. To further delineate the possible role and interactions of Notch with BCR-ABL in CD34+ primary cells from chronic-phase CML, we used P-crkl detection as a surrogate assay of BCR-ABL tyrosine kinase activity. Our data revealed that Imatinib (IM) induced BCR-ABL inhibition results in significant (p<0.05) upregulation of Notch activity, assessed by Hes1 expression. Similarly, inhibition of Notch leads to hyperactivation of BCR-ABL. This antagonistic relationship between Notch and BCR-ABL signalling was confirmed in K562 and ALL-SIL cell lines. In K562, we further validated this antagonistic relationship by inhibiting histone deacetylase (HDAC) - an effector pathway of Hes1, using valproic acid (VPA) - a HDAC inhibitor. Finally, we also confirmed the potential antagonism between Notch and BCR/ABL in In Vivo, using publically available GSE-database, by analysing gene expression profile of paired samples from chronic-phase CML patients pre- and post-Imatinib therapy. Thus, we have demonstrated an antagonistic relationship between Notch and BCR-ABL in CML. A combined inhibition of Notch and BCR-ABL may therefore provide superior clinical response over tyrosine-kinase inhibitor monotherapy by targeting both quiescent leukaemic stem cells and differentiated leukaemic cells and hence must be explored.

ObjectiveNotch signalling has been implicated in haematopoietic stem cell self-renewal. Although several studies have tested the effect of activating or inhibiting the Notch signalling pathway in stem cells, no study has yet determined the functional differences associated with expressing Notch1. The aims of this study were to characterise the expression of human cell-surface Notch1 in cord blood (CB) CD34(+) cells and to study the function of Notch in CD34(+) cells in vitro.MethodsA monoclonal antibody against the extracellular domain of Notch1 was developed, and Notch1 expression in CB CD34(+) cells was assessed by flow cytometry. CB CD34(+) cells were sorted on the basis of their Notch1 expression and cultured in serum-free media. Single sorted CD34(+)CD38(-)Notch1(+)/(-) cells were cultured for 8wks on murine stroma monolayers and assayed for stem cell activity and lineage potential using a cobblestone area-forming cell (CAFC) assay.ResultsCell-surface Notch1 expression was characterised in various primitive CD34(+) cell compartments including a small subpopulation of CD34(+)CD38(-) cells. We found the CD34(+)CD38(-)Notch1(+) population to be enriched for stem cell activity. Moreover, CD34(+)CD38(-)Notch1(+), but not Notch1(-) cells, demonstrated multilineage potential.ConclusionsThese data show that Notch1 is expressed on a functionally distinct subpopulation of CD34(+) cells that is highly enriched for stem cell activity and multilineage potential and could suggest that Notch1 could be used as a novel stem cell marker.

Recent studies have highlighted the role of Notch signalling in the development of T cell acute lymphoblasic leukaemia (T-ALL). Over-expression of Notch3 and gain of function mutations in the Notch1 gene have been reported. The aims of this study were to determine the effect of Notch signalling on apoptosis in human T-ALL cell lines and to identify targets of Notch signalling that may mediate this effect. Functional studies showed that inhibition of Notch signalling using gamma secretase inhibitors promoted glucocorticoid-induced apoptosis in cells carrying gain of function mutations in Notch1. Moreover, ectopic expression of constitutively activated Notch provided protection against glucocorticoid-induced apoptosis, indicating that signalling via Notch may also contribute to the development of T-ALL by conferring resistance to apoptosis. Microarray analysis revealed that GIMAP5, a gene coding for an anti-apoptotic intracellular protein, is upregulated by Notch in T-ALL cell lines. Knockdown of GIMAP5 expression using siRNA promoted glucocorticoid-induced apoptosis in T-ALL cells carrying gain of function mutations in Notch1 and in T-ALL cells engineered to express ectopic constitutively activated Notch indicating that Notch signalling protects T-ALL cells from apoptosis by upregulating the expression of GIMAP5.

Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1). Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

The hematon is a three-dimensional aggregate of cells which is able to produce all blood types. To be able to do this, it must be able to create within the cell aggregate a microenvironment which enables haematopoietic stem cell maintenance, renewal and differentiation. A first step was taken towards the creation of artificial hematopoietic stem cell microniches in vitro by the creation with dielectrophoresis of hemispherical cell aggregates of a height of 50-100 mum with a defined internal architecture similar to that of a putative hematon. It is shown that, after their dielectrophoretic manipulation, the cells remain viable and active. Cells within the aggregate are in direct contact with each other, potentially allowing direct cell-cell communication within the cell construct. Some cell immobilisation methods are explored for further stabilising the 3-D organisation of the cell aggregate after its formation. The introduction of traceable individual cells into the artificial microniche is demonstrated.

A high-speed cell sorter capable of a throughput speed 4-5-fold greater than commercially available systems was developed and evaluated as a processing tool for isolating purified hematopoietic stem cell grafts. The clinical high-speed sorter (CHSS) serves as a single-pass, multiparameter processing tool that provides the means to isolate a highly purified population of cells from starting cell populations with extremely low frequencies. The sorter incorporates environmental barriers to create a sterile environment for cell processing. Monoclonal antibodies and reagents produced under good manufacturing practices (GMP) are used to isolate hematopoietic stem cells by means of the CHSS. Using this technology, the CD34+Thy-1+Lin- hematopoietic stem cell population has been isolated from normal adult bone marrow and mobilized peripheral blood. The sorted cells have been shown to be sterile and viable and to retain hematopoietic function.

Notch signaling regulates diverse cell fate decisions during development and is reported to promote murine hematopoietic stem cell (HSC) self-renewal. The purpose of this study was to define the functional consequences of activating the Notch signaling pathway on self-renewal in human HSCs. Subsets of human umbilical cord blood CD34(+) cells were retrovirally transduced with the constitutively active human Notch 1 intracellular domain (N1ICD). N1ICD-transduced cells proliferated to a lesser extent in vitro than cells transduced with vector alone, and this was accompanied by a reduction in the percentage and absolute number of CD34(+) cell populations, including CD34(+)Thy(+)Lin(-) HSCs. Ectopic N1ICD expression inhibited cell cycle kinetics concurrent with an upregulation of p21 mRNA expression and induced apoptosis. Transduction of cells with HES-1, a known transcriptional target of Notch signaling and a mediator of Notch function, had no effect on HSC proliferation, indicating that the mechanism of the Notch-induced effect is HES-1-independent. The results of this study show that activation of the Notch signaling pathway has an inhibitory effect on the proliferation and survival of human hematopoietic CD34(+) cells populations. These findings have important implications for strategies aimed at promoting self-renewal of human HSCs.

Positive dielectrophoresis can be used to create aggregates of animal cells with 3D architectures. It is shown that the cells, when pulled together into an aggregate by positive dielectrophoresis in a low‐conductivity iso‐osmotic solution, adhere to each other. The adherence of the cells to each other is non‐specific and increases in time, and after 10–15 min becomes strong enough to immobilize the cells in the aggregate, enabling the ac electric field to be released, and the iso‐osmotic buffer to be replaced by growth or other media. Cell viability is maintained. The new method of immobilization significantly simplifies the construction of aggregates of animal cells by dielectrophoresis, and increases the utility of dielectrophoresis in tissue engineering and related areas. Biotechnol. Bioeng. 2007;98: 694–700. © 2007 Wiley Periodicals, Inc.

The formation of aggregates of mammalian cells at interdigitated oppositely castellated electrodes by positive dielectrophoresis was investigated. It is shown that, by using a constant small flow of fresh sorbitol iso-osmotic buffer through the chamber to remove ions leaking from the cells, a high positive DEP force can be maintained throughout the formation of the aggregates. Flow-rate dependent optima were found in the aggregate height as a function of the electrode size. It is shown that at low flow rates the creation of aggregates of mammalian cells with heights over 150 µm is feasible using relatively low voltages (20 Vpk–pk, 1 MHz). The formation of layered aggregates of two specialized cell types—stromal cells and Jurkat T lymphocytes—is demonstrated. The work confirms that dielectrophoresis can be reliably used for the formation of aggregates with three-dimensional architectures, which could be used as artificial microniches for the study of interactions between cells.

Objective: Notch signalling is known to promote hematopoietic stem cell self-renewal and to influence the lineage commitment decisions of progenitor cells. The purpose of this study was to investigate the mechanism of Notch-induced apoptosis in the erythroleukaemic cell line TF-1, and in primary cord blood CD34+ cells.Methods: Retroviral constructs containing constitutively active forms of Notch as well as components of the Notch signalling pathway were used to transduce cells and their effect on cell cycle kinetics and apoptosis assayed by immunostaining for the S-phase marker Ki67 and Annexin V.Results: We found that TF-1 cells undergo cell cycle arrest followed by apoptosis in a cytokine-independent manner in response to active Notch. Transduction of TF-1 cells with known targets of Notch signalling, Deltex1, HES1 and HERP2, showed that Notch-induced cell cycle arrest was not mediated by these proteins. However, analysis of cell cycle gene expression revealed that Notch signalling was associated with an up-regulation of IFI16 expression in TF-1 cells and in primary cord blood CD34+ cells.Conclusion: These data demonstrate that, in the context of TF-1 cells, Notch signalling can induce cell cycle arrest and apoptosis. (C) 2008 Elsevier Inc. All rights reserved.

Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate.