A role for chondroitin sulfate proteoglycan in regulating the survival and growth of neural stem cells

A ROLE FOR CHONDROITIN SULFATE PROTEOGLYCAN IN REGULATING THE SURVIVAL AND GROWTH OF NEURAL STEM CELLS THAM ANH VU MULY B.Sc.(Hon.), University of Nottingham, UK) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements This thesis would not have been possible without the help of many people First and foremost I would like to thank my supervisor Dr Sohail Ahmed for providing an opportunity for me to work in his lab, considering that I came to him in rather unusual circumstances He has been very supportive throughout my studies and gave me a great deal of freedom to explore and learn I would also like to thank my cosupervisor Dr Gavin Dawe for supporting this collaborative work I am eternally grateful to my postdoc, Dr Srinivas Ramasamy, for helping me with many experiments, particularly the clonal hydrogel assay and NCFCA But more importantly, he was a constant source of engaging scientific conversations, always challenging my thinking and had helped me focus a great deal in the latter part of my study Similarly, my fellow student, Gan Hui Theng, had also been a great source of support, encouragement and great ideas I would like to thank Srivats Hariharan for providing excellent microscopy support, but more importantly for the many engaging lunch time conversations that had made my research life a lot more fun I would also like to thank Dr Goh Wah Ing for taking the pain to read this thesis and correct my bad English Credits are also due to the countless people who have provided reagents and equipments throughout my studies Lastly, I would like to thank my family for being the pillar of strength in my life I would like to thank my parents for their contemporary thinking and the willingness to give me freedom from a young age I would like to thank my husband and my children who had supported my study without complaint, and for unconditional love even when I haven’t given them all the attention they deserved i Table of contents Acknowledgements .i Table of contents ii List of publications vi Summary vii List of Figures ix List of Tables xi List of Abbreviations .xii INTRODUCTION 1.1 Mammalian development 1.2 The stem cell concept 1.3 Symmetrical and asymmetrical division 1.4 Types of stem cells 1.4.1 Embryonic stem cells 1.4.2 Somatic stem cells 1.4.3 Stem cells and cancer 14 1.5 Neural development 16 1.6 Neural stem cells 19 1.6.1 Embryonic neural stem cells 20 1.6.2 Adult neural stem cells .24 1.6.3 Neural stem cell applications .26 1.7 Methods to study neural stem cells 28 1.7.1 Identifying neural stem cells in vivo 28 1.7.2 In vitro analysis – the neurosphere assay 35 1.8 The stem cell niche .38 1.8.1 The Notch pathway 39 1.8.2 The canonical Wnt pathway .41 1.8.3 The sonic hedgehog pathway .41 ii 1.8.4 Epidermal growth factor and fibroblast growth factor 42 1.8.4.1 1.8.5 1.9 EGFR signalling in neural stem cells 43 Neural stem cell conditioned medium 44 Proteoglycans 45 1.9.1 Heparan sulfate proteoglycans 48 1.9.2 Chondroitin sulfate proteoglycan .49 1.9.2.1 1.10 CSPG signalling mechanisms 51 Aims of current work 55 MATERIALS AND METHODS 56 2.1 Isolation of NSCs and the NSA 56 2.1.1 Clonal hydrogel culture 56 2.1.2 Adherent culture 57 2.2 NSC-Conditioned medium 57 2.3 CSPG and inhibitors on neurosphere formation and proliferation 58 2.4 ATP assay and estimation of population doubling time .59 2.5 Apoptosis and survival assays 59 2.6 Serial passaging 61 2.7 Differentiation 61 2.8 Immunohistochemistry 63 2.9 Neural colony forming cell assay (NCFCA) 64 2.10 Single neurosphere gene profiling 65 2.11 CSPG signalling .65 2.11.1 Chemical inhibitor studies 65 2.11.2 Western analysis 66 2.12 Cytokine array .67 RESULTS 68 3.1 NSC conditioned medium stimulates neurosphere formation .68 3.2 CSPG is responsible for the NSC-CM stimulation of neurosphere formation 70 3.3 CSPG is essential for neurosphere formation 71 3.3.1 Exogenous CSPG stimulates neurosphere formation 71 3.3.2 CSPG stimulates neurosphere formation in clonal assays 73 3.3.3 Stimulation of neurosphere formation is specific to CSPG 75 iii 3.3.4 Endogenous CSPG is essential for neurosphere formation 75 3.3.5 Glycosaminoglycan sulfation and neurosphere formation 79 3.4 CSPG is essential for neural precursor proliferation 82 3.4.1 Exogenous CSPG stimulates neural precursor proliferation .82 3.4.2 Endogenous CSPG is required for neural precursor proliferation .84 3.4.3 Inhibition of CSPG in adherent culture inhibit neural precursor proliferation 86 3.5 CSPG is essential for neural precursor survival 88 3.6 Characterisation of CSPG generated cells 91 3.6.1 CSPG and NSC self-renewal .93 3.6.2 CSPG and multipotency .95 3.6.3 Neural colony-forming cell assay 102 3.6.4 Genetic profiling of CSPG generated neurospheres 104 3.7 CSPG signalling .109 3.7.1 Chemical inhibitor studies 112 3.7.1.1 CSPG stimulates neurosphere formation via EGFR 112 3.7.1.2 CSPG stimulates neurosphere formation via PI3K/Akt 115 3.7.1.3 CSPG stimulates neurosphere formation via JAK/STAT 115 3.7.1.4 ERK is involved in neurosphere formation and proliferation 118 3.7.1.5 p38 MAPK inhibits neurosphere formation .120 3.7.1.6 Notch is involved in neurosphere formation and proliferation 122 3.7.1.7 Shh is involved in neurosphere formation and proliferation .125 3.7.1.8 Phosphatases are involved in neurosphere formation and proliferation 125 3.7.1.9 Wnt inhibits neurosphere formation 128 3.7.1.10 Rho/ROCK is involved in neurosphere formation 128 3.7.2 Biochemical analysis of CSPG signalling 131 3.7.2.1 CSPG upregulates EGFR and phospho-EGFR expression 131 3.7.2.2 CSPG increases phospho-STAT3 expression 135 3.7.2.3 CSPG increases Akt and phospho-Akt expression 137 3.7.2.4 CSPG does not affect ERK and phospho-ERK expression .139 3.7.2.5 CSPG does not affect p38 and phospho-p38 MAPK expression 139 3.7.2.6 CSPG and cell cycle proteins 142 iv 3.8 Other factors in conditioned medium 142 DISCUSSION 145 4.1 CSPG stimulates NSC survival .147 4.1.1 CSPG stimulates clonal neurosphere formation 147 4.1.2 CSPG promotes extensive self-renewal 148 4.1.3 CSPG increases the percentage of multipotent neurospheres 148 4.1.4 CSPG increases neural colony formation 149 4.1.5 Genetic profile of CSPG generated neurospheres .150 4.1.6 CSPG reduces apoptosis and stimulates neurosphere formation in the absence of EGF 151 4.2 Enumeration of NSC frequency .152 4.3 CSPG regulation of NSC survival verses NSC self-renewal 156 4.4 CSPG stimulates neural precursor proliferation 158 4.5 Role of endogenous CSPG .159 4.5.1 Neurosphere formation, proliferation and differentiation 160 4.5.2 CSPG maintains the neurosphere structure 162 4.6 CSPG structure and function 164 4.6.1 Protein verse glycosaminoglycan chains 164 4.6.2 Sulfation pattern and CSPG function .165 4.7 CSPG signalling .168 4.7.1 EGFR-related pathways mediate CSPG stimulation of neurosphere formation 169 4.7.2 Non-EGFR-related pathways 175 4.8 4.9 Implications of current work 179 Conclusion and future directions 182 Reference .184 v List of publications • Tham M, Ramasamy S, Gan H, Ramachandran A, Poonepalli A, Yu YH, Ahmed S Chondroitin sulfate proteoglycan stimulates neural stem cell survival via EGFR signalling pathways Manuscript in preparation • Ahmed S, Gan H, Lam CS, Poonepalli A, Ramasamy S, Tay Y, Tham M, Yu YH Transcription factors and neural stem cell self-renewal, growth and differentiation Cell Adh Migr 2009, 27; 3(4) • Murphy S, Krainock R, Tham M Neuregulin signaling via erbB receptor assemblies in the nervous system Mol Neurobiol 2002 Feb; 25(1):67-77 • Tham M, Sim M.K & Tang F.R Location of renin-angiotensin system components in the hypoglossal nucleus of the rat Regul Pep 2001 101: 51-57 • Richardson M, Braybrook C, Tham M, Moore GE and Stanier P Molecular cloning and characterization of a human laminin receptor psedogene in Xq21.3 Gene 1998 206: 145-150 • Abu-Hayyeh S, Eddleston J, Murdoch J, Tham M, Copp AJ and Stanier P Linkage mapping of Lims1, the murine homolog of the human LIM domain gene PINCH, to mouse chromosome 10 Cytogenet Cell Genet 1998 82: 46-48 Abstracts: • Tham AVM and Ahmed S CSPG is essential for neural stem cell survival and proliferation, for neurosphere formation and maintenance 6th Asia Pacific Symposium on Neuroregeneration (APSNR), Singapore 2008 • Doudney K, Eddleston J, Itani A, Tham M, Murdoch J, Copp A and Stanier P Construction of a PAC and P1 contig around the Lp critical region on mouse chromosome Mol Med Symp IC London 1999 • Doudney K, Eddleston, J, Tham M, Murdoch J, Paternotte C, Gregory S, Copp A and Stanier P Comparative mapping of the mouse and human homologous chromosome regions containing the mouse NTD mutant Lp locus Report of the 5th International Workshop on Human Chromosome Mapping Cytogenet Cell Genet 1999 87: 166 • Doudney K, Eddleston J, Braybrook C, Itani A, Tham M, Murdoch J, Copp A and Stanier P Transcript mapping in the Lp critical region on mouse chromosome 13th International Mouse Genome Conference, Philadelphia, 1999 vi Summary Neural stem cells (NSCs) give rise to the nervous system during development, and persist in the adult to replace neurons in certain regions of the brain NSCs can be isolated and maintained as neurospheres in vitro, and give rise to neurons, oligodendrocytes and astrocytes upon differentiation Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix and are involved in neural development Here I show that CSPG is a component of the NSC-conditioned medium (NSC-CM), and is partly responsible for the ability of NSC-CM to stimulate neurosphere formation Neurospheres can arise from NSCs or lineage restricted progenitors To determine whether CSPG stimulates NSCs or progenitors, two cardinal features of stem cells were evaluated, self-renewal and multipotency CSPG generated neurospheres can be expanded for at least seven times, and demonstrate increased proliferation in the neural colony forming cell assay (NCFCA) Clonallyderived neurospheres from CSPG treated cultures show increased multipotency CSPG generated neurospheres display similar genetic profile as controls The NSC frequency was estimated based on the percentage of clonally-derived neurospheres that displayed multipotency CSPG increases the NSC frequency by more than threefold Thus CSPG stimulates NSC survival CSPG also increases neurosphere size and reduces the population doubling time of neurospheres in culture, indicating that CSPG stimulates proliferation In addition, CSPG is involved in maintaining the 3dimensional structure of neurospheres Using chondroitinase-ABC, sodium chlorate, β-D-xyloside and differentially sulfated chondroitin sulfate glycosaminoglycans (CSGAGs), I dissected the structure of CSPG and attribute the regulation of NSC survival and proliferation to the full proteoglycan structure including specific sulfation motifs, vii whereas maintenance of the neurosphere structure requires only the CS-GAG Lastly, I demonstrate that CSPG functions in NSC survival and proliferation via EGFR, JAK/STAT3 and PI3K signalling pathways viii List of Figures Figure 1.1 Amnion structure and cell movements during human gastrulation Figure 1.2 Differentiation of human tissues Figure 1.3 Haematopoietic and stromal cell differentiation .11 Figure 1.4 Epidermal stem cell niche 13 Figure 1.5 Self-renewal signalling pathways in stem and cancer cells .15 Figure 1.6 Neurulation in the mammalian embryo 17 Figure 1.7 Regional specification of the developing brain 18 Figure 1.8 NSCs and their progeny in the developing forebrain 21 Figure 1.9 Lineage trees of neurogenesis 22 Figure 1.10 Polarized features of neuroepithelial cells, radial glial cells and basal progenitors 22 Figure 1.11 The SVZ niche, cell types and stem cell lineage 25 Figure 1.12 Neurogenesis in the adult rodent brain 29 Figure 1.13 Prospective isolation of stem cells 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