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Characterization of a Stem Cell Population Derived from Human Peripheral Blood and Its Therapeutic Potential in Brain Tumors

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Characterization of a Stem Cell Population Derived from Human Peripheral Blood and Its Therapeutic Potential in Brain Tumors Characterization of a stem cell population derived from human peripheral blood and its therapeutic potential in brain tumors A thesis submitted to Imperial College London for the degree of Doctor of Philosophy in the Faculty of Medicine Catherine T. Flores, BSc, MSc Department of Haematology, Faculty of Medicine, Division of Investigative Science, Imperial College London, Hammersmith Hospital, London Supervisors Professor Myrtle Y Gordon Department of Haematology Faculty of Medicine Imperial College London Hammersmith Campus London W120NN Dr Deanna L Taylor Department of Neurosciences Faculty of Medicine Imperial College London Hammersmith Campus London W120NN 1 Abstract Adherent CD34 + cells are a stem cell enriched population with a high frequency of primitive stem cells that are genetically primed to differentiate into tissue-specific lineages. These cells make up the adherent CD34 + cells in peripheral blood which are the focus of this study. The first aim of this study was to transcriptionally characterize adherent CD34+ cells relative to non- adherent CD34 + cells using a whole human genome Affymetrix U133 plus 2 array. The subsequent analysis demonstrated transcriptional differences in genes which are involved in quiescence, cell cycle, homing and adhesion, which are likely to be relevant to the suitability of adherent CD34 + cells for clinical application. To verify the microarray analysis, a selection of representative transcripts was chosen and their relative expression was compared using real-time qPCR. To analyze further the stem cell characteristics of adherent CD34 + cells, the DNA replication timing kinetics of pluripotency-associated genes in bone marrow-derived adherent CD34+ cells, non-adherent CD34+ cells, and foetal mesenchymal stem cells were conducted and compared to those of embryonic stem (ES) cells. This showed that pluripotency-associated genes Oct4 and Nanog replicate early in the cell cycle similar to ES cells. The second aim of this study was to examine the potential use of adherent CD34+ cells in neural injury. As adherent CD34+ cells are primitive stem cells, it was reasonable to hypothesize that these cells would differentiate into neural cells in vitro , but these experiments revealed that these cells are not neurogenic and, in our hands, do not differentiate into neural cells. The experiments revealed that adherent CD34+ cells differentiate efficiently and reproducibly into microglia-like cells (CD34MG cells). CD34MG cells upregulated microglia-associated antigens and demonstrated functional characteristics of microglia cells including TNF α and IL-6 release in response to lipopolysaccharide. CD34MG also stimulate responders in a mixed lymphocyte reaction, suggesting antigen presenting capabilities. To determine if these cells might be exploited for use in brain tumour therapy, it was shown that CD34MG migrate and invade in response to brain tumour glioma cells. Results also suggest that these cells are capable of presenting glioma specific antigen to immune cells in an autologous lymphocyte reaction in vitro . The combined results suggest that CD34MG cells might be exploited for therapeutic purposes in brain tumours. Stem cells are currently being explored for their therapeutic potential. This study demonstrates that adherent CD34+ cells and CD34MG cells may be useful for clinical use. 2 Declaration of originality I declare that this is the result of my own work carried out between 2005 and 2008 in the Department of Haematology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN. All the work, analysis, and illustrations were performed by me, unless otherwise specified in the text. Catherine Flores 3 Table of Contents Abstract ................................................................................................................................ 2 Declaration of originality ...................................................................................................... 3 Table of Contents.................................................................................................................. 4 Figures.................................................................................................................................. 7 Tables ................................................................................................................................... 7 Abbreviations........................................................................................................................ 8 Acknowledgements…………………………………………………………………………...10 Chapter 1 Introduction ........................................................................................................ 11 1.1 Stem cells.................................................................................................................. 11 1.2 Stem cell division...................................................................................................... 12 1.3 Stem cell multiplicity ................................................................................................ 15 1.4 Bone marrow stem cell niche..................................................................................... 17 1.4.1 Developmental migration.................................................................................... 18 1.4.2 Haematopoietic inductive microenvironment ...................................................... 18 1.4.3 Multilineage priming .......................................................................................... 19 1.5 Stem cell DNA replication timing.............................................................................. 20 1.6 The nervous system................................................................................................... 21 1.6.1 Injury in the CNS................................................................................................ 23 1.6.2 Bone marrow infiltrates in neural injury.............................................................. 23 1.6.3 Endogenous neurogenesis in injury..................................................................... 25 1.6.4 Extra-cranial sources of stem cell for repair ........................................................ 25 1.7 G-CSF mobilization................................................................................................... 28 1.8 Stem cell therapy ...................................................................................................... 28 1.9 Glioblastoma Multiforme .......................................................................................... 32 1.10 Aims ………………………………………………………………………………….34 Chapter 2 Materials and methods ........................................................................................ 35 2.1 Source of material for study....................................................................................... 35 2.1.1 Leukaphereses .................................................................................................... 35 2.1.2 Foetal human mesenchymal stem cells and human fibroblast cells...................... 35 2.1.3 Neural stem cells ................................................................................................ 35 2.1.4 Glioblastoma cell lines........................................................................................ 36 2.1.5 N9 microglia cell line ......................................................................................... 36 2.1.6 Embryonic stem cells……………………………………………………………...37 2.2 Isolation of CD34 + cells............................................................................................. 36 2.3 Isolation of plastic adherent CD34+ cells................................................................... 37 2.4 Neural Differentiation................................................................................................ 37 2.4.1 Neuronal induction protocol 1............................................................................. 37 2.4.2 Neuronal induction protocol 2............................................................................. 37 2.4.3 Neuronal indiction protocol 3 ............................................................................. 38 2.4.4 Neuronal induction protocol 4............................................................................. 38 2.4.5 Neuronal induction protocol 5............................................................................. 38 2.4.6 Neuronal induction protocol 6............................................................................. 38 2.4.7 Neuronal induction protocol 7............................................................................. 39 2.4.8 Neuronal induction protocol 8............................................................................. 39 2.4.9 Neuronal induction protocol 9............................................................................. 39 2.5 Microglial differentiation........................................................................................... 39 2.5.1 Astrocyte conditioned medium ........................................................................... 39 2.6 RNA Isolation ........................................................................................................... 40 2.6.1 RNA salt precipitation ........................................................................................ 41 4 2.6.2 Agilent 2100 Bioanalyser, RNA 6000 Pico/Nano Assay ....................................
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