Roles of long non coding RNAs in human embryonic stem cell pluripotency and neural differentiation 1

ROLES OF LONG NON-CODING RNAS IN HUMAN
EMBRYONIC STEM CELL PLURIPOTENCY AND
NEURAL DIFFERENTIATION





NG SHI YAN
B. Sc (Honors)
National University of Singapore, 2008





A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND
ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2012

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Acknowledgements

I would like to express my deepest gratitude to the many people who made this thesis
possible. I thank my supervisor Dr. Lawrence Stanton for his invaluable guidance,
advice, support and his belief in me. His mentorship encouraged independence,
creativity, and allowed me the freedom to grow and develop. I have learnt a lot during

the past four years, which has been an extremely enriching and inspiring experience. I
would also like to thank my thesis advisory committee, Dr. Wang Hongyan, Dr. Paul
Robson, and Dr. Gerald Udolph, for their critical feedback along the way.

I am especially grateful to Dr. Rory Johnson, who introduced me to the world
of lncRNAs. I am thankful for the insightful discussions we had, the custom lncRNA
microarray which he designed, as well as his feedback and comments. I also thank
Gireesh Bogu, for the RNA-seq analyses. Dr. Irene Aksoy was most generous in
providing me with a human iPS cell line. I am also thankful for stimulating
discussions with Dr. Akshay Bhinge. It is also a pleasure to thank everyone in the GIS
Stem Cell groups for companionship, support and helpful discussions.

Most importantly, my family has given me incredible support and
encouragement. Their love and understanding helped me overcome the obstacles in
the process. My parents have provided me with an incredible environment to grow
and nurture; my sister was amazingly supportive, understanding and a great
companion. I also thank my fiancé for his patience and companionship, who made
long hours in the lab more pleasant.
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Table of contents

Acknowledgements ii
Table of contents iii
Abstract vii
List of figures ix
List of tables xiii
Abbreviations xiv
Chapter I: Introduction ………………………………………………………… 1
1.1 Transcriptional control of stem cell pluripotency 1

1.1.1 The transcription factors OCT4, NANOG and SOX2 constitute the human embryonic
stem cell transcriptional core 1
1.1.2 An expanded transcriptional regulatory circuit maintaining pluripotency 2
1.1.3 Non-coding RNAs modulate pluripotency by regulating expression of the core
transcription factors and/or downstream genes 6
1.2 Directed neural differentiation of hESCs 7
1.2.1 Neural induction – lessons from the embryo 7
1.2.1.1 Inhibiting the TGF-β signaling pathway enhances neural induction 8
1.2.1.2 Stromal co-culture induces neural conversion 10
1.2.2 Regional specification of midbrain dopamine neurons 11
1.2.3 Radial glia cells are neuronal progenitors in vivo 14
1.3 Long non-coding RNAs in biology 15
1.3.1 Long non-coding RNAs in pluripotency 16
1.3.2 Long non-coding RNAs in neural development 19
1.3.2.1 Nkx2.2AS 19
1.3.2.2 Evf2 20
1.3.2.3 Malat1 20
1.4 Molecular mechanisms of long non-coding RNA function 21
1.4.1 LncRNAs behave as scaffolds that target protein complexes to specific genomic loci
to regulate gene transcription 23
1.4.2 LncRNAs with enhancer-like functions 24
1.4.3 LncRNAs regulate gene expression by behaving as competing endogenous RNAs or
promoting mRNA decay 24
1.4.4 Other molecular functions of lncRNAs 25

Chapter II: Aims and Objectives ……………………………………………… 26
2.1 Main goals of the thesis 26
2.2 Thesis outline 27

Chapter III: Materials and Methods …………………………………………… 28

3.1 Feeder-free culture of human pluripotent stem cells 28
3.2 Expansion and mitotic inactivation of MEF cells 28
3.3 Preparation of MEF-conditioned medium 29
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3.4 Culture of PA6 mouse skull bone marrow stromal cells 29
3.5 Differentiation of hESCs into neural progenitors and dopaminergic
neurons 30
3.5.1 Co-culture of PA6 and hESCs for stromal-derived inducing activity (SDIA) 30
3.5.2 Isolation and expansion of neural progenitor cells (NPCs) 30
3.5.3 Differentiation of NPCs into dopaminergic (DA) neurons 31
3.6 Culture of human fetal mesencephalon-derived neural stem cells (ReN-
VM) 32
3.6.1 Maintenance of ReN-VM cells 32
3.6.2 Differentiation of ReN-VM cells 32
3.7 Small-interfering RNA (siRNA)-mediated gene silencing 33
3.7.1 Transfection of H1 hESCs with siRNAs 33
3.7.2 Transfection of ReN-VM cells with siRNAs 33
3.8 Stably transfected hESC lines 34
3.9 RNA extraction 35
3.9.1 Extraction of total RNA 35
3.9.2 Extraction of nuclear and cytoplasmic RNA (RNA fractionation) 35
3.10 Reverse transcription of RNA to cDNA 36
3.11 Quantitative real-time PCR (qPCR) 36
3.12 Analysis of gene expression by microarray 39
3.12.1 RNA amplification for Illumina bead chips 39
3.12.2 Illumina bead chip hybridization 40
3.12.3 RNA amplification and hybridization on custom designed Agilent arrays 40
3.12.4 Statistical analysis of microarray data 40
3.13 RNA-sequencing (RNA-seq) 41

3.13.1 RNA-seq library preparation 41
3.13.2 RNA-seq data analysis 42
3.14 Western blot 43
3.15 Immunocytochemistry 44
3.16 Fluorescence-activated cell sorting (FACS) 45
3.17 Co-immunoprecipitation (co-IP) 46
3.17.1 co-IP with overexpression constructus 46
3.17.2 Endogenous co-IP 46
3.18 Chromatin immunoprecipitation (ChIP) 47
3.19 RNA immunoprecipitation (RIP) 48
3.20 Biotinylated RNA pulldown 49
3.21 RNA fluorescence in situ hybridization 50

Chapter IV: Neural Differentiation of Human Pluripotent Stem Cells ……… 52
4.1 Introduction 52
4.2 Results 53
4.2.1 A homogenous population of neural progenitors was derived from hESCs by the
modified SDIA method 53
4.2.2 Human ESC-derived neural progenitors differentiated into functional dopamine
neurons with high efficiency 60
4.3 Discussion 67
4.3.1 Stromal induction induced neuronal differentiation from pluripotent stem cells with
high efficiency 67
4.2.3 High efficiency neuronal differentiation by chemically defined methods 67
4.4 Conclusion 68

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Chapter V: Identification of Long Non-coding RNAs Associated with
Pluripotency and Neural Differentiation ……………………………………… 70

5.1 Introduction 70
5.2 Results 72
5.2.1 Microarray expression profiling identifies differentially expressed lncRNAs 72
5.2.2 Identification of lncRNAs associated with pluripotency (pluripotent lncRNAs) 74
5.2.3 Identification of lncRNAs associated with neural progenitors (NPC lncRNAs) 77
5.2.4 Identification of lncRNAs associated with neuronal differentiation (neuronal
lncRNAs) 79
5.3 Discussion 81
5.4 Conclusion 82

Chapter VI: Long Non-coding RNAs Regulate Human Embryonic Stem Cell
Pluripotency ……………………………………………………………………… 83
6.1 Introduction 83
6.2 Results 84
6.2.1 Screening for possibly functional pluripotent lncRNAs 84
6.2.2 Pluripotent lncRNAs are regulated by transcription factors 91
6.2.3 Knockdown of lncRNAs result in hESC differentiation 94
6.2.4 Pluripotent lncRNAs physically associate with SUZ12 and SOX2 98
6.3 Discussion 101
6.3.1 LncRNAs join the pluripotency alliance 101
6.3.2 LncRNAs possibly function as a modular scaffold 101
6.4 Conclusion 104

Chapter VII: Long Non-coding RNAs are Indispensable in Neurogenesis 105
7.1 Introduction 105
7.2 Results 107
7.2.1 Screening for possibly functional neuronal lncRNAs 107
7.2.2 Neuronal lncRNAs are required for neuronal differentiation 112
7.2.3 Neuronal lncRNAs support neurogenesis by associating with nuclear proteins 118
7.2.4 Cytoplasmic lncRNA_N2 affects microRNA expression 121

7.3 Discussion 122
7.3.1 Neuronal lncRNAs act via diverse mechanisms 122
7.3.2 Neuronal lncRNAs form part of a repressive complex to silence glia genes 122
7.4 Conclusion 124

Chapter VIII: Brain lncRNA RMST Regulates Neurogenesis by Association with
SOX2 ……………………………………………………………………………… 125
8.1 Introduction 125
8.2 Results 127
8.2.1 RMST is highly expressed in the human brain and upregulated during neurogenesis
127
8.2.2 RMST is developmentally regulated by transcription factor REST 129
8.2.3 RMST is indispensable for neurogenesis, but not required for maintenance of neuronal
identity 132
8.2.4 Nuclear-retained RMST physically associates with RNA-binding protein
hnRNPA2B1 and transcription factor SOX2 134
8.2.5 RMST and SOX2 co-regulate a common pool of genes 140
8.2.6 RMST does not regulate SOX2 expression 144
8.3 Discussion 145
8.3.1 RMST forms part of a complex that is required for neurogenesis 145
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8.3.2 RMST may change the binding patterns of SOX2 to chromatin 146
8.3.3 RMST may bind to proteins other than hnRNPA2B1 and SOX2 147
8.4 Conclusion 147

Chapter IX: Conclusion and Perspectives ……………………………………… 149
9.1 Overall conclusions 149
9.1.1 Identification of functional human lncRNAs 149
9.1.2 LncRNAs specific to hESCs maintain the pluripotent state 150

9.1.3 Neuronal lncRNAs support neurogenesis by associating with transcription factors 150
9.2 Limitations and future work 151
9.2.1 Discovery of novel lncRNAs 151
9.2.2 Epigenetic regulation of pluripotency 151
9.2.3 RMST modulation SOX2 activity 152
9.2.4 SOX2 and lncRNA association 152
9.2.5 Long non-coding RNAs or short peptides 153
9.3 Concluding remarks 153

References ………………………………………………………………………… 155

Appendices ……………………………………………………………………… 169
Appendix I: List of 152 genes downregulated upon RMST and SOX2 knockdown 169
Appendix II: List of 331 genes upregulated upon RMST and SOX2 knockdown 170












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Abstract

Long non-coding RNAs (lncRNAs) are a recently discovered class of transcripts
encoded within the human genome. LncRNAs have been proposed to be key
regulators of biological processes, including stem cell pluripotency and neurogenesis.
However, at present very little functional characterization of lncRNAs involved in
differentiation has been carried out in human cells. In this thesis, functional
characterization of lncRNAs in human development is addressed using human
embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal
differentiation. Human ESCs were robustly and efficiently differentiated into neurons,
and expression of lncRNAs was profiled using a custom-designed microarray. Some
hESC-specific lncRNAs involved in pluripotency maintenance were identified, and
shown to physically interact with SOX2, and PRC2 complex component, SUZ12.
Using a similar approach, we identified lncRNAs required for neurogenesis.
Knockdown studies indicated that loss of any of these lncRNAs blocked
neurogenesis, and immunoprecipitation studies revealed physical association with
REST and SUZ12.

In particular, a neuronal lncRNA, RMST, was found to be essential for
neurogenesis. Knockdown of RMST in human neural stem cells prevented
neurogenesis. RNA pulldown and RNA immunoprecipitation indicated that RMST
physically associated with the RNA-binding protein hnRNPA2B1 and the
transcription factor SOX2. Perturbation studies, followed by genome-wide
transcriptional profiling indicated that RMST and SOX2 co-regulate a large pool of
targets. Interestingly, knockdown of RMST resulted in reduced SOX2 occupancy at its
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target gene promoters, suggesting that RMST may alter SOX2 binding to chromatin
during neurogenesis. Together, this study represents important evidence for an
indispensable role of lncRNAs in human brain development.

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List of figures

Figure 1.1 OCT4, SOX2 and NANOG form the core transcription factors
governing pluripotency of hESCs. 3

Figure 1.2 An extended ES transcriptional network and regulatory circuit. 5

Figure 1.3 Patterning of the neural tube generates unique domains for neuronal

progenitors. 13

Figure 1.4 The differentiation of pluripotent stem cells into neuroepithelial stem
cells and radial glia. 15

Figure 1.5 Correlation of expression profiles of lncRNAs with protein-coding
gene markers during embryoid body (EB) differentiation. 17

Figure 1.6 A model for lincRNA integration into the molecular circuitry of
embryonic stem cells. 18

Figure 1.7 Paradigms for how lncRNAs function at the molecular level. 22

Figure 4.1 Stromal co-culture of H1 hESCs resulted in neural differentiation. 55

Figure 4.2 Differentiation of hESCs into a monolayer neural progenitor
population. 57

Figure 4.3 Neural progenitors derived from H1 hESCs (H1-NPCs) homogenously
expressed neural stem cell and radial glia markers. 59

Figure 4.4 Schematic representation of the differentiation of hESC-derived radial
glia-like NPCs into midbrain dopaminergic (DA) neurons. 61

Figure 4.5 Immunofluorescence characterization of H1-derived neurons indicating
their midbrain dopaminergic identity. 62

Figure 4.6 Quantitative PCR (qPCR) analysis confirming midbrain dopaminergic
identity. 63

Figure 4.7 Flow cytometry analysis showed that H1-NPCs differentiated into TH
+

midbrain dopamine neurons with high efficiency. 65

Figure 4.8 Dopamine enzyme-linked immunosorbent assay indicated that H1-
derived dopamine neurons were responsive to depolarization. 66

Figure 5.1 Microarray expression profilinf identified diferentially expressed
lncRNAs during neural differentiation of hESCs. 74

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Figure 5.2 Identification of lncRNAs important in pluripotency, neural induction,
and neuronal differentiation. 75

Figure 6.1 Three lncRNAs were exclusively expressed in hESCs and iPSCs. 87

Figure 6.2 Pluripotent lnRNAs are low abundance transcripts. 88

Figure 6.3 RNA-seq analysis of pluripotent lncRNAs in H1 hESCs, indicating
transcriptional start and end sites. 90

Figure 6.4 Schematic showing OCT4 and NANOG binding sites in the vicinity of
the lncRNAs. 92

Figure 6.5 Pluripotent lncRNAs are possibly regulated by OCT4 and NANOG.
93

Figure 6.6 Pluripotent lncRNAs can be effectively targeted by siRNAs. 94

Figure 6.7 Knockdown of pluripotent lncRNAs resulted in hESC differentiation.
95

Figure 6.8 Knockdown of pluripotent lncRNAs resulted in loss of OCT4. 96

Figure 6.9 Microarray analysis indicated that knockdown of pluripotent lncRNAs
caused hESC differentiation. 97

Figure 6.10 Pluripotent lncRNAs are preferentially localized in the nucleus. 98

Figure 6.11 Nuclear pluripotency lncRNAs physically associated with PRC2
component SUZ12, and the pluripotent transcription factor SOX2. 100

Figure 6.12 Proposed mechanism for role of lncRNA in hESC pluripotency. 102

Figure 6.13 In silico prediction of lncRNA-protein interactions supporting the
proposed mechanism of lncRNAs functioning as a modular scaffold for
chromatin modifiers and transcription factors. 103

Figure 7.1 In situ hybridization images of lncRNA expression showing that
lncRNAs were specifically localized to the specific brain regions. 106

Figure 7.2 RNA-seq analysis indicating transcription start and end sites of
neuronal lncRNAs. 110

Figure 7.3 Tissue specificity of the neuronal lncRNAs RMST, lncRNA_N1,
lncRNA_N2 and lncRNA_N3. 111

Figure 7.4 Relative abundance of neuronal lncRNAs, compared to that of GAPDH

mRNA levels. 112

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Figure 7.5 Schematic representation of differentiation of ReN-VM neural stem
cells following transfection of siRNAs. 113

Figure 7.6 Neuronal lncRNAs can be efficiently targeted by siRNAs. 114

Figure 7.7 Knockdown of neuronal lncRNAs prevent neurogenesis. 116

Figure 7.8 Loss of TUJ1
+
cells upon knockdown of neuronal lncRNAs. 117

Figure 7.9 Knockdown of neuronal lncRNAs resulted in cells adopting a glia fate.
117

Figure 7.10 Apart from lncRNA_N2, the other neuronal lncRNAs were nuclear-
localized. 119

Figure 7.11 RNA immunoprecipitation indicated that lncRNA_N1 and lncRNA_N3
associated with SUZ12 and REST. 120

Figure 7.12 Quantification of changes in hosted miRNAs in response to
lncRNA_N2 knockdown. 121

Figure 7.13 Neuronal lncRNAs possibly act in trans. 123

Figure 8.1 Rmst is highly expressed in the midbrain and prospective dopamine

neurons. 126

Figure 8.2 Expression of RMST in somatic tissues measured by qPCR. 128

Figure 8.3 RMST expression was upregulated during neurogenesis. 129

Figure 8.4 ENCODE ChIP-seq database indicates the presence of a REST binding
site upstream of RMST. 130

Figure 8.5 ChIP-PCR indicating REST occupancy upstream of the lncRNA
RMST. 131

Figure 8.6 RMST expression was regulated by transcription factor REST. 131

Figure 8.7 Knockdown of RMST prevented neurogenesis. 132

Figure 8.8 Overexpression of RMST enhanced neuronal differentiation of ReNVM
cells. 133

Figure 8.9 Loss of RMST in neurons had no apparent effect on cellular
morphology. 134

Figure 8.10 No significant changes in expression of neuronal markers upon
knockdown of RMST in neurons. 134

Figure 8.11 RMST is a nuclear-localized lncRNA. 135
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Figure 8.12 Biotinylated RMST pulldown, coupled to LC-MS/MS mass

spectrometry identified hnRNPA2B1 and SOX2 as protein partners of
the lncRNA. 137

Figure 8.13 Western blot confirmed that hnRNPA2B1 and SOX2 specifically
interact with RMST. 138

Figure 8.14 RNA immunoprecipitation (RIP) established in vivo binding of RMST
with hnRNPA2 and SOX2. 138

Figure 8.15 Co-immunoprecipitation (Co-IP) of hnRNPA2-FLAG and SOX2 in the
absence of RNA. 139

Figure 8.16 Proposed model of the RMST complex. 140

Figure 8.17 Knockdown of components of the RMST complex prevented
neurogenesis. 141

Figure 8.18 RMST and SOX2 regulate a common pool of targets. 143

Figure 8.19 Transcript levels of SOX2 remained unchanged following RMST
knockdown. 145

Figure 8.20 Depletion of RMST resulted in decreased SOX2 occupancy at target
genes. 146

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List of tables

Table 3.1 Sequences of siRNAs used. 34

Table 3.2 List of primers for protein-coding genes and Universal Library Probe
(Roche) number used for qPCR. 37

Table 3.3 List of primers used for lonc non-coding RNAs in qPCR. 39

Table 3.4 Primary antibodies and respective dilutions used in Western blot. 44

Table 3.5 Primary antibodies and respective dilutions used in
immunofluorescence (IF) and flow cytometry (FACS). 45

Table 3.6 Sequences of primers used for ChIP-PCR. 48

Table 5.1 Genes expressed in H1-derived neurons were highly enriched for Gene
Ontology (GO) terms relating to neuronal differentiation. 73

Table 5.2 List of the 36 pluripotency lncRNAs. 76

Table 5.3 List of the 24 NPC lncRNAs. 78

Table 5.4 List of the 35 neuronal lncRNAs. 80

Table 6.1 List of pluripotent lncRNAs that occupy a unique location in the
genome, and can be targeted by RNAi. 86

Table 6.2 Pluripotent lncRNAs in this study. 89

Table 7.1 List of 25 neuronal lncRNAs that occupied a unique location in the
human genome, and can be targeted by RNAi. 108

Table 7.2 Neuronal lncRNAs in this study. 109

Table 8.1 Table summarizing microarray findings upon knockdown of RMST or
SOX2 in neural stem cells. 143

Table 8.2 Gene Ontology (GO) analysis of the 152 genes in the si-RMST and si-
SOX2 overlap. 144

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Abbreviations

µg Micrograms
µl Microlitre
bFGF Basic fibroblast growth factor
(FGF2)
BSA Bovine serum albumin
cDNA Complementary DNA
ChIP Chromatin
immunoprecipitation
DAPI 4’,6-diamidino-2-phenylindole
DMEM Dulbecco’s modified eagle’s
medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide
triphosphate
DTT Dithiothreitol
EDTA Ethylene Diamine Tetra-acetic
Acid
EB Embryoid body

ES cells Embryonic stem cells
FACS Fluorescence activated cell
sorting
FBS Fetal bovine serum
FDR False discovery rate
GFP Green fluorescent protein
GO Gene ontology
hnRNP heterogenous nuclear
ribonucleoprotein
IgG Immunoglobulin G
LiCl Lithium chloride
MAP2 Microtubule-associated protein
2
MEF Mouse embryonic fibroblast
OCT4 Octamer-binding transcription
factor 4
PAGE Polyacrylamide gel
electrophoresis
PBS Phosphate buffered saline
PCR Polymerase chain reaction
qPCR Quantitative PCR
RISC RNA-induced silencing
complex
RNA Ribonucleic acid
RNAi RNA interference
SDS Sodium dodecyl sulfate
siRNA Small interfering RNA
SOX SRY-related HMG Box
TBST Tris-buffered saline/ Tween-20

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Chapter I – Introduction

1.1 Transcriptional control of stem cell pluripotency

Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts, and
can be maintained undifferentiated in culture. Pluripotency and self-renewal are
hallmarks of embryonic stem cells (ESCs). Pluripotency, or the undifferentiated ESC
state that can give rise to mature cells of the three germ layers, is in part maintained
by an intricate interplay between transcription factors and their genomic targets.
Forced expression of the right cocktail of transcription factors is also known to induce
pluripotency in adult somatic cells (Takahashi and Yamanaka, 2006), further
demonstrating the pivotal role of transcription factors in the maintaining and inducing
the pluripotent cell state.

1.1.1 The transcription factors OCT4, NANOG and SOX2 constitute the human
embryonic stem cell transcriptional core
The transcription factors OCT4, NANOG and SOX2 play essential roles in early
embryonic development, and are among the pluripotency-associated factors that
maintain ESCs (Boiani and Scholer, 2005; Chambers et al., 2003; Niwa et al., 2000).
Genome-wide chromatin immunoprecipitation (ChIP) studies aimed at elucidating
transcription factor binding sites and regulation of pluripotency have led to the
discovery of a transcriptional regulatory circuitry in hESCs (Boyer et al., 2005;
Ivanova et al., 2006; Loh et al., 2006; Rao and Orkin, 2006). Numerous target genes
co-bound and co-regulated by OCT4, SOX2 and NANOG have been identified
(Figure 1.1A). In addition, genes that are co-bound by OCT4, NANOG and SOX2
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include those that promote pluripotency and self-renewal, such as OCT4, SOX2,

NANOG, STAT3, ZIC3, and components of the TGF-β and Wnt signaling pathways.
These observations suggested that OCT4, SOX2 and NANOG promote pluripotency
by positively regulating their own expression and genes encoding components of key
signaling pathways (Figure 1.1B).

Apart from binding to pluripotency targets, OCT4, SOX2 and NANOG co-
bound targets were also enriched for genes implicated in developmental processes.
These included genes that specify transcription factors important for differentiation
into the extra-embryonic, endodermal, mesodermal, and ectodermal lineages (Figure
1.1C). The observation that OCT4, SOX2 and NANOG co-occupy a set of repressed
genes that are key to developmental processes suggested that in addition to activating
pluripotency-associated genes, they also repress genes associated with differentiation.

1.1.2 An expanded transcriptional regulatory circuit maintaining pluripotency
In a landmark report by Takahashi and Yamanaka (2006), it was demonstrated that
pluripotency could be induced in somatic cells such as fibroblasts by the forced
expression of four factors, namely Oct4, Sox2, Klf4 and c-Myc. The induced
pluripotent stem cells (iPSCs) were similar to ESCs in morphology, proliferation,
surface antigens, telomerase activity, gene expression and epigenetic marks
(Takahashi et al., 2007). Surprising, they observed that Nanog was dispensable for the
reprogramming of fibroblasts into iPSCs, implying that other transcription factors,
such as Klf4, were also part of an extended regulatory network governing
pluripotency (Figure 1.2).

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Figure 1.1: (Adapted from Boyer et al., 2005) OCT4, SOX2 and NANOG form

the core transcription factors governing pluripotency of hESCs. (A) Venn
diagram representing the extent of overlap of OCT4, SOX2 and NANOG promoter
bound regions. (B) The interconnected autoregulatory loop formed by OCT4, SOX2,
and NANOG. Regulators are represented as blue circles while gene promoters are
shown as red rectangles. OCT4 and SOX2 are known to physically interact. (C) A
model for the core transcriptional regulatory network in hESCs, whereby OCT4,
SOX2 and NANOG are the core transcription factors that co-activate pluripotency-
associated factors, and co-repress differentiation genes.



From the iPSC studies, it was evident that Kruppel-like factor 4 (Klf4) was an
important component of the ES circuitry. It was a mysterious player among the four
“Yamanaka factors”, since there were no apparent defects in a loss-of-function assay.
It was later discovered that other Klf members, Klf2 and Klf5, could compensate the
loss of Klf4 in maintaining pluripotency, and depletion of all three Klf members led to
differentiation. Genome-wide ChIP assays revealed that these Klfs share many
common downstream targets of Nanog, and a NANOG promoter luciferase reporter
assay established that KLF4 directly regulates NANOG expression (Chan et al., 2009;
Jiang et al., 2008). This indicates that the core KLF circuit is integrated into the
NANOG transcriptional network, to specify gene expression unique to ESCs.

Several other transcription factors are also integral members of the ES
transcriptional regulatory network, including Dax1, Zfp281, Rex1 and Esrrb (Figure
1.2A; (Kim et al., 2008). Another example is Zfp206 or Zscan10, which encodes a
zinc finger transcription factor specifically expressed in pluripotent stem cells, and is
required for self-renewal of undifferentiated cells. Zfp206 physically associates with
both Oct4 and Sox2, and genome-wide mapping of Zfp206 binding sites in ESCs
revealed that Zfp206 targets were also co-regulated by Oct4 and Sox2, indicating that
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Zfp206 is also a key component of the regulatory network that maintains ESCs (Yu et
al., 2009).



Figure 1.2: (Adapted from Kim et al., 2008) An extended ES transcriptional
network and regulatory circuit. (A) A transcriptional regulatory circuit maintaining
pluripotency in ESCs, with five factors (Nanog, Oct4, Sox2, Dax1, and Klf4) showing
an auto-regulatory mechanism. (B) Transcriptional regulatory circuit within the four
Yamanaka reprogramming factors and their integration into the Nanog circuitry.




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1.1.3 Non-coding RNAs modulate pluripotency by regulating expression of the
core transcription factors and/or downstream genes
Non-coding RNAs (NcRNAs) are an important class of regulatory molecules that are
changing our concept of gene regulation. In particular, microRNAs (miRNAs) are
well-characterized regulators of gene expression. MiRNAs are short, approximately
22-nucleotide RNAs that attenuate gene expression post-transcriptionally by base-
pairing with target mRNAs which has a sequence in the 3’ untranslated region (UTR)
that imperfectly matches the six- to eight-nucleotide “seed sequence” of the miRNA.
The miRNA then inhibits translation or cause degradation of the mRNA by the RISC
complex when there is perfect complementarity with the seed sequence (Brodersen et
al., 2008; Farazi et al., 2008; Filipowicz et al., 2008), a phenomenon known as RNAi.

Recent reports have identified miRNAs that were upregulated in

differentiating ESCs and played a critical role in the control of pluripotency genes
(Judson et al., 2009; Suh et al., 2004; Tay et al., 2008a; Tay et al., 2008b; Xu et al.,
2009). miR-296, miR-470 and miR-134 were found to be up-regulated during retinoic
acid induced differentiation of mESCs, and were computationally predicted to target
the protein-coding sequences (CDS) of the pluripotent core transcription factors Oct4,
Sox2 and Nanog. Forced expression of the miRNAs led to ESC differentiation, as
well as down-regulation of Oct4, Nanog and Sox2 at both the mRNA and protein
levels (Tay et al., 2008a).

More recently, it was demonstrated that the forced expression of miRNAs
alone could reprogram adult somatic cells to attain pluripotency (Anokye-Danso et
al., 2011). The miR302/367 cluster specifically expressed in undifferentiated ESCs
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and iPSCs has been shown to be a direct target of Oct4 and Sox2, two of the most
important pluripotency transcription factors that are also required for iPSC
reprogramming. The authors also reported that miR367 expression was required for
reprogramming and activated Oct4 gene expression. Together, studies on miRNAs in
pluripotency point to an important role of miRNAs in regulating the transcriptional
network of pluripotency.

In recent years, long non-coding RNAs (lncRNAs) became prominent in the
research of ncRNAs, in part fueled by whole genomic and transcriptomic analyses
and deep sequencing technologies (Chen et al., 2011; Guttman et al., 2009; Lin et al.,
2011). LncRNAs are defined as RNA transcripts that are longer than 200 nucleotides,
and have little or no protein coding capacity. In contrast to the small ncRNAs such as
miRNAs, which are highly conserved and silence gene expression through specific
base-pairing with target mRNAs, lncRNAs are poorly conserved and regulate gene
expression by diverse mechanisms that are not entirely understood. The roles of
lncRNAs in pluripotency will be described in Section 1.3.

1.2 Directed neural differentiation of hESCs

1.2.1 Neural induction – lessons from the embryo
One of the characteristics of pluripotent stem cells is the ability to differentiate into
mature cell types of the three germ layers: ectoderm (including neural lineages),
mesoderm and the endoderm. However, the efficient differentiation of pluripotent
stem cells into specific neural cell types requires exposure of the cells to the right
culture conditions (an optimal concentration of growth factors, signaling molecules,
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inhibitors, and cell-cell signaling) for the right duration of time. Development of the
nervous system can be divided roughly into three processes: neural induction,
neurulation, and regional specification. Cues taken from the embryo during each of
these processes would be useful in establishing efficient methods for in vitro neural
differentiation.

Explant studies in the Xenopus had shown that the Spemann organizer could
induce a complete new dorsal axis, resulting in a twinned embryo with two distinct
heads. The Spemann organizer is involved in the induction of neural tissue from the
ectoderm by secreting bone morphogenetic protein (BMP) antagonists such as
chordin, noggin and cereberus. BMP4 is pivotal in neural induction, as it inhibits cells
from forming neural tissue (Finley et al., 1999). Therefore, inhibition of BMP
signaling is crucial for initiating neural differentiation. However, double knockout of
noggin and chordin in zebrafish did not prevent a neural plate from developing,
although there were defects in the neural tube. This suggests that other signaling
pathways, in addition to the inhibition of the BMP signaling, could be involved in
neural development. With insights from in vivo neural development, methods aimed
at efficient neural induction were reported, and described below.

1.2.1.1 Inhibiting the TGF-β signaling pathway enhances neural induction
The transforming growth factor beta (TGF-β) signaling pathway is involved in many
cellular processes, including cellular differentiation. The TGF-β superfamily of
ligands includes the BMPs, growth and differentiation factors (GDFs), anti-mullerian
hormone (AMH), Activin, Nodal and the TGFβs. The TGF-β superfamily ligands
bind to a type II receptor, which sets off a phosphorylating cascade culminating in the
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eventual phosphorylation of receptor-regulated SMADs. The SMAD complexes then
accumulate in the nucleus where they act as transcription factors that regulate gene
expression of their targets.

Noggin is a secreted factor expressed in the Spemann organizer. In Xenopus,
the Spemann organizer induces neural tissue from dorsal ectoderm and dorsalizes
lateral and ventral mesoderm. Noggin binds and inactivates the BMPs, and the
inhibition of the BMP signaling pathway in the ectoderm is the hallmark of neural
fate acquisition (Munoz-Sanjuan and Brivanlou, 2002). By recapitulating neural
development, Lamb et al. (1993) and Valenzuela et al. (1995) showed that Noggin
directly induced neural tissue, and was an endogenous neural inducer. Subsequently,
recombinant Noggin has been used in several hESC neural induction protocols
(Chambers et al., 2009; Elkabetz et al., 2008; Lee et al., 2007).

Recently, Dorsomorphin or Compound C was discovered to be a small
molecule substitute for recombinant Noggin (Yu et al., 2008; Zhou et al., 2010).
Dorsomorphin is an inhibitor of BMP signaling, identified in a screen for compounds
that perturb the dorsoventral axis formation in zebrafish, and selectively inhibits the
BMP type I receptors ALK2, ALK3 and ALK6 (Yu et al., 2008). This eventually
results in blockade of SMAD1/ 5/ 8 phosphorylation, and induces neural conversion
of hESCs by suppressing endoderm, mesoderm and trophectodoerm differentiation
(Morizane et al., 2011; Zhou et al., 2010).

In addition, the small molecule SB431542 was shown to enhance neural
induction by inhibiting the Lefty/ Activin/ TGF-β pathways by blocking
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phosphorylation of the ALK4, ALK5 and ALK7 receptors. The combined blockade of
the TGF-β pathway using SB431542 in combination with Noggin or Dorsomorphin
was reported to achieve very efficient neural induction (Chambers et al., 2009;
Morizane et al., 2011).

1.2.1.2 Stromal co-culture induces neural conversion
It was discovered by Kawasaki and colleagues (2000) that the co-culture of ES cells
and the mouse PA6 stromal cells could induce efficient dopamine neuronal
differentiation. They described the neuralizing activity conferred by the stromal cells
as “stromal cell-derived inducing activity” or SDIA. Since then, research groups have
attempted to unravel the basis of SDIA for efficient neural differentiation of hESCs
(Swistowska et al., 2010; Vazin et al., 2009; Vazin et al., 2008). To this end, Vazin et
al. (2008) found that PA6 cell surface activity was required for neural differentiation
of hESCs, as the PA6-conditioned medium (PA6-CM) was ineffective in neural
induction. In addition, it was reported that PA6-CM could induce dopaminergic
differentiation in neural progenitors derived from hESCs, but not directly from
hESCs, indicating that soluble factors, including Sonic hedgehog (SHH) secreted by
the PA6 stromal cells act on neural precursor cells to specify a dopaminergic fate
(Swistowska et al., 2010). SHH is a ligand of the Hedgehog signaling pathway, and a
ventralizing signal required for the development of midbrain dopamine neurons.

In attempts to chemically define SDIA, a genome-wide expression analysis
was performed to compare global gene expression differences between PA6 cells, and
various cell lines lacking the SDIA effect. Among the factors highly expressed by
PA6 cells, Vazin et al. (2009) discovered that the combination of stromal cell-derived

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factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor (IGF2) and
ephrin B1 (EFNB1) induced undifferentiated hESCs to dopamine neurons, mimicking
the effects of SDIA.

1.2.2 Regional specification of midbrain dopamine neurons
After neuroectoderm induction, the embryo undergoes neurulation, in which the
epithelial neural plate begins to furrow and the neural tube forms, which will
eventually differentiate into the spinal cord and the brain, forming the central nervous
system. Following neural tube closure, patterning is important for determining
regional cell fate, and this is mediated by signaling between adjacent cells by cell-
surface proteins, and by gradients of signaling molecules. The neural tube becomes
regionalized along the antero-posterior (AP) axis, as well as the dorso-ventral (DV)
axis.

Cell fate decisions along the DV and AP axes are dictated by factors from
signaling and organizing centers (Rowitch and Kriegstein, 2010). The formation of
midbrain (mesencephalic) dopamine neurons is directed by diffusible signals from the
notochord, floor plate, and isthmic organizer. Sonic hedgehog (SHH), secreted by the
notochord and floor plate, and fibroblast growth factor 8 (FGF8) secreted by the
isthmic organizer, are key molecules involved in dopaminergic differentiation (Ye et
al., 1998).

SHH is secreted initially by the notochord, then the floor plate, and
antagonizes the BMPs secreted by the roof plate, thereby creating a SHH gradient
along the DV axis (Figure 1.3A). In response to SHH signaling, several homeodomain