HUMAN EMBRYONIC STEM CELL-DERIVED NEURAL STEM CELLS: DERIVATION, DIFFERENTIATION AND MICRORNA REGULATION KWANG WEI XIN TIMOTHY NATIONAL UNIVERSITY OF SINGAPORE 2013 HUMAN EMBRYONIC STEM CELL-DERIVED NEURAL STEM CELLS: DERIVATION, DIFFERENTIATION AND MICRORNA REGULATION KWANG WEI XIN TIMOTHY (B.Sc (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declara ation declare that this thesis is my origin work and it has bee written by me in nal d en y I hereby d its entirety I have duly acknowled y dged all the sources of informatio n which hav been e f ve used in the thesis e This thesis has also not been su s n ubmitted for any degree in any uniiversity prev r e viously _ Kw wang Wei Xin Timothy X 24 Januar 2013 ry i Acknowledgements Firstly, I would like to express my gratitude to my supervisor, A/P Wang Shu, for his support and guidance through my PhD stint His encouragement, patience and instruction whilst allowing me to conduct my research independently has made this journey a valuable experience I thank my fellow lab-mates, past and present, for their assistance in my research work and for making this journey a less arduous and more enjoyable one; Mohammad Shahbazi, Dang Hoang Lam, Yovita Ida Purwanti, Jiakai Lin, Chrishan Ramachandra, Yukti Choudhury, Detu Zhu, Kai Ye, Esther Lee, Ghayathri Balasundaram, Poonam Balani, Seong Loong Lo, Chunxiao Wu, Jieming Zeng, and Ying Zhao I would like to express my sincere thanks to my family, especially my parents for their unceasing support and provision in all my endeavors I am also grateful to all my friends for their prayers and encouragement through this season I would like to thank the Institute of Bioengineering and Nanotechnology (IBN) for the resources and support granted to me to carry out my research work I also thank NUS Graduate School of Science and Engineering (NGS) for this opportunity to a PhD Additionally, I would also like to appreciate the NGS administrative staff for ever being ready to address my queries and to alleviate the administrative challenges that I may have encountered Most of all I would like to thank God for His abundant favor “He makes me lie down in green pastures He leads me beside still waters.” – Psalm 23:2 ii Table of Contents Summary vi  List of Tables viii  List of Figures ix  List of Abbreviations xi  Chapter 1: Introduction 1  1.1  Neural stem cells 1  1.1.1  A brief history of NSCs 1  1.1.2  The “definition” of an NSC 2  1.1.3  NSCs in development and regenerative medicine 4  1.1.4  NSCs in cancer therapy 6  1.1.5  Sources of human NSCs 8  1.1.5.1 1.1.5.2 1.2  Fetal and adult NSCs Pluripotent stem cell-derived NSCs Factors regulating self-renewal and differentiation of NSCs 11  1.2.1  1.2.1.1 1.2.1.2 Transcription factors 12  PAX6 13 SOX2 15 1.2.2  Extracellular factors 19  1.2.3  Epigenetic factors 21  1.3  MicroRNAs 22  1.3.1  Overview 22  1.3.2  Biogenesis 23  1.3.3  Mechanism of action 26  1.3.3.1 1.3.3.2 1.3.4  1.4  Target recognition 26 Target gene silencing 27 miRNAs in NSC self-renewal and fate commitment 30  Aims and objectives 32  Chapter 2: Materials and Methods 35  iii 2.1  Cell culture 35  2.2  Generation of NSCs from hESCs 35  2.3  Generation of GPCs from hESC-derived NSCs 37  2.4  Terminal differentiation of hESC-derived NSCs into glial cells and neurons 38  2.5  Immunocytochemistry 38  2.6  RNA isolation 39  2.7  Reverse transcriptase-PCR and Quantitative RT-PCR of mRNA 39  2.8  Flow cytometry 41  2.9  Western blot analysis 41  2.10  In vitro migration assay 43  2.11  MicroRNA microarray 43  2.12  MicroRNA target prediction 44  2.13  Quantitative RT-PCR of miRNA 45  2.14  miRNA mimics, miRNA expression vectors and transfection 46  2.15  Luciferase reporter assay 47  2.16  Baculovirus preparation and transduction 49  2.17  Statistical analysis 51  Chapter 3: Derivation of neural progenitors from hESCs 52  3.1  Introduction and aims 52  3.1.1  Deriving NSCs 52  3.1.2  Deriving GPCs 53  3.2  Results 55  3.2.1  NSCs derived from hESCs via neurosphere culture display neuroectodermal identity 55  3.2.2  NSCs derived from hESCs via neural rosette formation display neuroectodermal identity 59  iv 3.2.3  Downregulation of early neuroectodermal genes and upregulation of GPC markers during differentiation from NSCs to GPCs 61  3.2.4  In vitro glioma-tropic properties of genetically modified hESC-derived NSCs and GPCs 67  3.3  Discussion 73  3.3.1  hESC-derived NSCs 73  3.3.2  hESC-derived GPCs 75  3.3.3  Future work 78  Chapter 4: Regulation of neuroectodermal genes by miRNA 80  4.1  Introduction and aims 80  4.2  Results 82  4.2.1  miR-22, -21, -221 and -145 are upregulated in GPCs compared to NSCs and are predicted to target PAX6 mRNA 82  4.2.2  PAX6 is a prospective target of miR-22 and miR-221 88  4.2.3  miR-145 is predicted to target SOX2 94  4.2.4  SOX2 is downregulated by miR-145 in NSCs 95  4.3  Discussion 105  4.3.1  Future work 108  Chapter 5: Summary and conclusion 110  References 113  Appendices 131    v Summary Over the last two decades there is burgeoning interest in neural stem/progenitor cells (NSCs) for both developmental research and cell-based therapeutic applications Although functional properties of human NSCs, in terms of their tumor-homing, in vivo regenerative and in vitro differentiation capacities, have been extensively studied, the molecular mechanisms underlying their self-renewal and differentiation are incompletely, if not poorly, understood Human embryonic stem cells (hESCs) offer a valuable source of NSCs to elucidate these mechanisms Here, we derived NSCs from hESCs and further differentiated these hESC-derived NSCs into NG2+ glial progenitor cells (GPCs) PAX6 and SOX2 are two transcription factors that characterize NSCs, and function as key determinants of the human neuroectodermal fate that drive neurogenesis or self-renewal, respectively Accordingly, we observed the downregulation of PAX6 and SOX2 expression in hESC-derived NSCs upon differentiation into GPCs microRNAs (miRNAs) are negative regulators of gene expression that have reportedly been implicated in NSC self-renewal and fate commitment, and thus are plausibly involved in the downregulation of PAX6 and SOX2 in NSCs during differentiation towards the glial lineage Utilizing miRNA microarrays, we have identified four miRNAs, miR-21, -22, -145 and -221, to be upregulated in GPCs compared with NSCs, among which miR-22 and miR-221 were demonstrated to be putative PAX6-targeting miRNAs The ectopic expression of miR145 by baculoviral vectors repressed SOX2 protein expression in human NSCs, while inhibition of miR-145 using baculoviral decoy vectors induced the opposite Thus, this study extends upon previous findings that miR-145 regulates SOX2 expression in hESCs and glioma cells, and indicates that miR-145 modulates the transition from multipotency to 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(TPM1) The Journal of biological chemistry 282, 14328-14336 Relative expression Appendices 1.2 0.8 0.6 0.4 0.2 NS GPC 7-days post-diff (7.2) (7.4) (5.8) (3.2) (81) (156) miR-145 miR 21 Hela (3.3) U87 (7.3) (4.2) miR-22 (2.8) (11.8) (2.2) miR-221 Appendix Figure A1 Relative endogenous expression levels of miR-145, -21, -22 and -221 in HeLa and U87 cells Real-time qPCR analysis of miR-22, -21, -221 and -145 expression in hESC-derived neurosphere NSCs (NS), GPCs days post-differentiation from hESC-derived neurosphere NSCs, HeLa and U87 Values in ( ) represent relative fold decrease in expression normalized to 5S rRNA and expressed relative to GPC Data represents mean + SD of triplicates Xu, N., et al (2009) Cell 137: 647-658 Appendix Figure A2 Validation of miR-145 targeting of the OCT4, SOX2, and KLF4 3′ UTRs Luciferase reporter assay where HeLa cells were transfected with luciferase reporter constructs, which carry wildtype (WT) or mutant 3’ UTRs of OCT4, SOX2 and KLF4, along with miR-145 precursor mimics Mutant UTRs have a bp deletion in the miR-145 target site miR-145 specifically represses its targets Image is from Figure of Xu et al (2009) 131 Xu, N., et al (2009) Cell 137: 647-658 Appendix Figure A3 Effect of miR-145 on endogenous OCT4, KLF4, and SOX2 in hESCs (B–C) Relative mRNA (B) and protein (C) levels of OCT4, SOX2, and KLF4 in control Lenti-scr or Lenti-miR-145 transduced hESCs (E–F) Relative mRNA (E) and protein (F) levels of OCT4, SOX2, and KLF4 in LNA-scr or LNA-miR-145 transfected hESCs In real-time RT-PCR, GAPDH mRNA is the normalization control (B and E) In western blot (C and F), protein level quantification was normalized to GAPDH Image is from Figure of Xu et al (2009) PAX6 0.9 0.9 0.9 1.0 1.1 1.0 1.1 1.1 0.9 1.2 1.0 1.0 SOX2 ReN GAPDH Appendix Figure A4 Effect of miR-21, -22, -221 and -145 on PAX6 and SOX2 expression in ReNcell NSCs Western blot analysis of PAX6 and SOX2 in ReNcell NSCs 72 hours after transfection with miR-21, miR-22, miR-221, miR-145, or negative control mimics Densitometry was performed using ImageJ software and protein amounts are normalized to β-actin loading control and presented relative to miRNA mimic negative control 132 .. .HUMAN EMBRYONIC STEM CELL- DERIVED NEURAL STEM CELLS: DERIVATION, DIFFERENTIATION AND MICRORNA REGULATION KWANG WEI XIN TIMOTHY (B.Sc (Hons), NUS)... mechanisms of regulation of NSC differentiation 1.1.5.2 Pluripotent stem cell- derived NSCs Human embryonic stem cells (hESCs), which are pluripotent cells derived from the inner cell mass of blastocyst-stage... of Human Embryonic Stem Cells in mTeSRTM1” (STEMCELL Technologies Inc.) In brief, H1 hESCs were maintained on BD matrigel hESC-qualified matrix (BD Biosciences) with mTeSRTM1 medium (STEMCELL