The laboratory is interested in purifying normal and leukaemic human stem cells using the non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation as a read out assay. Comparison of the phenotype, and molecular pathways regulating especially self-renewal are the focus of the group. Other projects are aimed at understanding the interactions between normal and leukaemic stem cells with their microenvironnment. All these projects should shed light into pathways or interactions that are more specifically used by LSC and where therapeutic intervention might be developed.
Leukaemic stem cell
Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukaemia initiating cells. Recently we showed that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukaemia cells. We showed 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 leukaemia samples, contains all or at least most leukaemia initiating cells capacity (Taussig et al., 2008; Blood 112: 568-75). Based on these results we examined both the phenotype of the LSC in different patient groups and also determine the frequency of LSC using a more immuno-deficient NSG mouse models (Taussig et al., submitted to Blood; Vargaftig et al., in preparation).
Leukaemic stem cells and their microenvironment
Over the years, we have been trying to purify LSCs and test their repopulating activity using our xenotransplantation model. It appears that like normal HSC, leukaemia might be dependent on microenvironment clues. Thus, we have developed a way using two-photon microscopy to visualise and describe the location and nature of this niche. To our surprise, we showed that both calvaria and long bones are highly vascularised and thus the notion of a separate vascular and osteoblastic niche should be revisited (Lassailly et al., in preparation). New studies are aiming now at describing the composition of the niche in both normal and leukaemic situation (Figure 1) and deciphering the composition of the haematopoietic stem cell niche.
Figure 1. Intravital microscopy of live bone marrow for non invasive observation of human leukemic cells in vitro.
Status of the stroma in AML patients
We investigate the status of the stroma in AML patients. For this we have studied the establishment of MSC from AML bone marrow samples. The proliferation and differentiation capacity as well as their secretome have been investigated. We showed that in most cases where confluent mesenchymal stem cells can be established, these cells proliferate much slower than their normal counterpart and also after a few passages they disappeared. We demonstrated that this decrease in proliferation was associated with an increase in senescence based on the presence of the biomarker beta-galactosidase and also an increase in p16. Despite this decrease in proliferation, the stroma from AML patients secreted a lot of cytokines. At present we are investigating what the function of these senescent stroma on AML proliferation and normal cells inhibition are (F. Afonso et al., In preparation).
Studying LSC in vitro
As described previously, Acute myeloid leukaemia (AML) is characterised by an invasion of the bone marrow by leukaemic myeloid blasts arrested at various maturation steps. Studies of AML have shown that only a small fraction of AML cells have the capacity to initiate leukaemia (SCID Leukemia Initiating Cells - SL-IC) when injected into severe combined immunodeficient mice. SL-ICs are the only experimental representation of Leukaemic Stem Cells. Because the AML xenograft model is prospectively a blind and long experiment with no or limited monitoring techniques and has a requirement for high cell numbers with approximately 30% of patient samples failing to engraft recipients, one of the goals in the Haemopoietic Stem Cell laboratory is to set up of an in vitro culture system to maintain LSCs. Preliminary studies indicated that with the use of different stroma supporting cells it is possible to maintain LSC in vitro for at least three weeks.
Role of mutated CEBPa in human haematopoiesis
C/EBPa (CEBPA) is mutated in approximately 8% of AML in both familial and sporadic AML and, with FLT3 and NPM1, has received most attention as a predictive marker of outcome in patients with normal karyotype disease. Mutations clustering to either the N- or C-terminal (N- and C-ter) portions of the protein have different consequences on the protein function. In familial cases the N-ter form is inherited with patients exhibiting long latency periods before the onset of overt disease, typically with the acquisition of a C-ter mutation. Despite the essential insights, murine models provide the functional consequences of wild-type CEBPa in human haematopoiesis and how different mutations are involved in AML development have received less attention. Our data underlines the critical role of C/EBPa in human haematopoiesis and demonstrates that C/EBPa mutations are insufficient to convert normal human haematopoietic stem/progenitors (HSC/HPCs) into leukaemic initiating cells, although individually each altered normal haematopoiesis. It provides the first insight into the effects of N- and Cterminal mutations acting alone, into the combined effects of N/C double mutants, the potential relevance of NC-ter mutations. Our results mimicked closely what happens in CEBPA mutated patients and contrasts to cebpa murine models (Bustamante-Quintana et al., In preparation).
For a list of refereed research papers, see Publications (in navigation on left).
