Blood, Stem Cell and Cancer

Introduction

The Blood, Stem Cell and Cancer Research Group focuses on clinical application of translational research into bone marrow derived stem cell and blood diseases. The group applies 'the state of art' molecular, biochemical and cell culture techniques on clinical samples to dissect out the complex mechanisms controlling normal stem cell and conditions leading to the formation of cancer stem cell. Knowledge gained from these laboratory results is then applied to improve patient outcomes. Our group has three research strands: stem cell transplantation, malignant haematology and haemostasis.

Stem Cell Transplantation

Multi-potent differentiation potential of human BM-derived mesenchymal stem cells

The aim of this project is to evaluate the ability of bone marrow (BM) derived mesenchymal stem cells (MSCs) to produce mature functioning cells of non-bone marrow tissues. We have taken a multi-disciplinary approach to enhance our ability to success by collaborating with researchers in other scientific areas. We have established a culture system and a panel of bio-markers for studying differentiation of BM MSCs into cells of mesodermal and ectodermal lineages. Using specific stimuli, we have shown BM MSCs can differentiate into intervertebral disc-like cells or with a different culture system to early neuronal cells. The BMP pathway has been identified to be critical in the discogenic differentiation of BM MSC. We are now examining the differentiation fate of BM MSC in normal and degenerative intervertebral discs in in vivo animal models in collaboration with Dr A Diwan's research team at St George Hospital Sydney. The success of driving BM MSCs to early neuronal fate has led us to transplanting these cells into the 6-OHDA unilaterally lesioned Parkinsonian rat model. The in vivo studies were conducted in collaboration with the research team of Professor Alan Mackay-Sim, Griffith University, QLD. So far, we found short-term survival of grafted cells and the presence of an inflammatory response at the graft site. The absence of early neuronal markers at the graft site also suggests that the transplanted human MSCs did not elicit endogenous neuronal differentiation in the host brain. Studies are underway to examine the nature of the immune response at the graft site.

Improving donor CD34+ stem cells to reconstitute T cell compartment in adult haematopoietic stem cell transplants

Haematopoietic Stem cell transplantation (SCT) from allogeneic (non-self) donors is a well-recognised and effective treatment for malignant and non-malignant haematological diseases. Impaired immune reconstitution, graft versus host disease (GVHD) and graft versus tumour effect (GVT) are the major hurdles to improving patient survival. We hypothesise that stem cell quality and thymic function are key factors in the effective immune reconstitution post SCT. The intended clinical application of this research is to identify therapies, which can selectively enhance immune reconstitution in individual BM transplants. We have used a stromal cell co-culture system to study the generation of T cells from haematopoietic stem cells. Our work has shown that the Delta/Notch signalling system initiates T cell differentiation but this signalling pathway needs to be switched off to allow further T cell differentiation. Further, we have shown that CD7 signalling is important in T cell differentiation in both early and late stages. We are currently characterizing gene expression in Delta/Notch and CD7 signallings. Running in parallel to the laboratory research, we are tracking these early T lymphoid progenitors, T cell subsets and their correlations with immune reconstitution, GVHD and GVT in patients undergoing allogeneic SCT in the pioneering program at St Vincent's Hospital, which has performed well over 1000 stem cell transplants since its inception in 1975.

Malignant Haematology

Identification of drug resistance genes by microarray profiling of leukaemic cells treated with tyrosine kinase inhibitors in Philadelphia positive acute lymphoblastic leukaemia (Ph+ ALL)

The cure rate of Ph+ALL remains low in spite of recent advances in the treatment of leukaemia. Imatinib and dasatinib, tyrosine kinase inhibitors, is highly successful in treating Ph+ chronic myeloid leukaemia but is ineffective against Ph+ ALL. This project comprises of gene expression profiling of leukaemic cells from patients receiving imatinib by microarray with downstream protein function validation and aims to dissect novel processes involved in imatinib resistance which may enable the design of new drugs to combat this cancer. Of > 400 genes with altered expression identified, six candidate genes were selected for further analysis. The expression changes of four of the six candidate genes were also observed in Ph+ cell lines treated with dasatinib, a second-generation tyrosine kinase inhibitor. We identified one of these six genes, Protein Kinase C epsilon (PKCε), a positive regulator of survival, proliferation, as the potential gene responsible for drug resistance. We are examining if this kinase also participates in drug resistance in dasatinib, a second generation tyrosine kinase inhibitor. Thus, inhibition of PKCε in Ph+ ALL may represent a novel way to improve the effectiveness of these tyrosine kinase inhibitors.

Role of microRNAs in the pathogenesis of acute myeloid leukaemia

In the last 10 years, microRNAs have been shown to be important in gene regulation and in 2004, the loss of microRNAs was shown to contribute to the development of chronic lymphocytic leukaemia. We have hypothesised that microRNA abnormalities may be involved in the pathogenesis of acute myeloid leukaemia with normal karyotype (NK-AML). Data obtained from microRNA microarray analysis of samples from patients with NK-AML were correlated with clinical information. Bioinformatic analysis was done in collaboration with researchers at the Department of Mathematics, University of Sydney. A number of microRNAs that targets against known oncogenes or tumour suppressor genes were identified. We are currently performing experiments to verify if the targets of these microRNAs and the role of these differentially expressed microRNAs in in vitro AML models with respect to proliferation, apoptosis and differentiation.

Haemostasis and Thrombosis

Evaluation of the procoagulant properties of platelets and microparticles in malignant and non-malignant states

Microparticles (MP) are vesicular fragments of the plasma membrane released upon cellular activation and/or apoptosis. These microparticles possess negatively charged phospholipids such as phosphatidylserine on their outer surface, allowing for the binding of activated coagulation factors X and V and the subsequent activation of the clotting pathway. The investigation of the role of microparticles in the pathogenesis of diseases including cancer and their complications is an emerging field. Our group has demonstrated the procoagulant property of MP using a newly developed clotting assay and MP functional assay based on flow cytometry. Recently we have also identified a sub-population of MP that do not bind Annexin V and possess little procoagulant activity. The focus of our research is aimed at discovering clinical significance of MP in cancer and bleeding disorders.

In vitro and in vivo study of the procoagulant and anticoagulant effects of detergent sclerosants.

Modern sclerotherapy used for the treatment of varicose veins is performed using detergent sclerosants, to destroy the endothelium of the target veins leading to complete vessel fibrosis. Little is known however about the effects of these sclerosants on haemostasis. A significant, albeit uncommon morbidity associated with sclerotherapy is deep vein thrombosis and pulmonary embolism. The aim of this project is to assess the effects of these sclerosants on the coagulation and fibrinolytic pathways of the haemostatic system. Data generated to date has provided new knowledge on haemostatic effects of sclerosants routinely used in sclerotherapy.