I have a PhD in Molecular Hematology, during which I looked at the role of microRNAs and small extracellular vesicles in chemotherapy-induced bystander effects. In this project, I explored the cross-talk between mesenchymal stem and haematopoietic stem cells in a bone marrow microenvironment model.
Cytotoxicity and cell viability assessments are very important parameters that are widely used in fundamental research and drug development to determine the safety profile of toxic compounds. These assays measure the degree to which a substance can cause toxic damage to cells or cell death. There are different assays that have been employed to determine the cytotoxicity of substances. These assays either determine enzymatic function, cell viability, mitochondrial activity, lipid metabolism, cell proliferation and/or cell death. These assays entail use of different kinds of dyes such as trypan blue exclusion dye, neutral red, acridine orange and propidium iodide to stain the cells. Trypan blue dye permeates compromised cell membrane to stain necrotic cells. However, this can lead to false positive and false negative results as it does not provide information on sub-lethal injury. As a result, neutral red and acridine orange can be used as counterstains for trypan blue to stain the lysosome of live cells. Acridine orange can also be used to stain nucleic acids in living cells and is usually co-stained with propidium iodide or ethidium bromide. This is because propidium iodide and ethidium bromide permeate only compromised plasma membrane thus co-staining cells with these dyes can provide vital information that can be used to differentiate between live and dead cells.
Part of the book: Cytotoxicity
Haematopoiesis is a tightly regulated process, by intrinsic and extrinsic factors, to produce lifelong blood cell lineages within the bone marrow. In the bone marrow microenvironment, mesenchymal stem cells and haematopoietic stem cells play important roles to ensure that haematopoiesis is maintained. These cells contain purines and pyrimidines that control intercellular process such as energy transport. However, in some cases, this process may be misregulated thus leading to the production of various diseases, including leukaemia. As a result, bone marrow cells may be stimulated via stress or induced hypoxia, and this leads to the release of purine and pyrimidine nucleotides and nucleosides into the extracellular space, and activation of autocrine/paracrine feedback loops. These extracellular nucleotides and nucleosides, and their respective cell surface receptors are involved in purinergic signaling that control different physiologic functions in cells including proliferation, differentiation, and cell death. These extracellular nucleotides and nucleosides include ATP, UTP, adenosine diphosphate (ADP), UDP and adenosine however the most important players are ATP and its metabolite adenosine. ATP is degraded via a sequential activity of ectonucleotidases. ATP, adenosine and these ectonucleotidases play very important roles in the tumour microenvironment crucial to disease development, progression, and aggressiveness by modulating immune response to leukaemia treatment and increasing homing of leukaemic cells.
Part of the book: Purinergic System
Stromal cells are stem cells in the bone marrow microenvironment that can ‘talk’ with neighbouring and distant cells within the bone marrow microenvironment. Stromal cells propagate this intercellular communication via cytokines, growth factors as well as small extracellular vesicles. The interaction between stromal cells and the haematopoietic stem cells, is crucial in the regulation of haematopoiesis. Aberration in this regulatory process will lead to the development of various diseases, including cancer. These stromal cells also play important role in the patient’s response to cancer therapy. As a result, these stromal cells may be crucial in the development and metastasis of cancer within the bone marrow microenvironment. In this chapter, we will explore the role of these stromal cells in carcinogenesis and cancer metastasis.
Part of the book: Cancer Metastasis