Induced Pluripotent Stem Cells as an Alternative to Embryonic Stem Cells for the Treatment of Type

362 Pages·2017·17.22 MB·English

Induced Pluripotent Stem Cells as an
Alternative to Embryonic Stem Cells
for the Treatment of Type 1 Diabetes

A thesis submitted by
Natalie Francis

For the degree of Doctor of Philosophy from Imperial College
London

Beta Cell Physiology Group
Department of Medicine

Imperial College London
Acknowledgements: Firstly, huge thanks to my NIBSC supervisor Chris, for all your help
and support throughout my PhD. Thank you for staying positive even when experiments
weren’t working! Thanks to all my colleagues at NIBSC, especially to Mel for all your
practical help, and to my colleagues at the UK Stem Cell Bank for passing on their
knowledge (and cells!) when I needed them. Thanks also to my supervisor Guy at Imperial
College for your helpful advice. Finally, thank you to my husband Rob, who proved that
getting married during a PhD is not always a bad idea, and for your continuous love and
support.

Declaration of originality: I declare that all work presented in this thesis is my own unless
otherwise referenced.

Copyright declaration: The copyright of this thesis rests with the author and is made
available under a Creative Commons Attribution Non-Commercial No Derivatives licence.
Researchers are free to copy, distribute or transmit the thesis on the condition that they
attribute it, that they do not use it for commercial purposes and that they do not alter,
transform or build upon it. For any reuse or redistribution, researchers must make clear to
others the licence terms of this work.
Abstract

Type 1 diabetes mellitus (T1DM) results from auto-immune destruction of the insulin-
secreting β-cells of the pancreas. The most common treatment is injection of exogenous
insulin, but this allows only partial control over blood glucose levels, so other therapies are
needed. Pancreatic islet transplantation has shown proof of principle for cell replacement
therapy to treat T1DM. There are several sources of cells which could be used, but much of
the focus has been on pluripotent stem cells, which are able to self-renew indefinitely in
culture and give rise to any cell in the body. Insulin-expressing cells have successfully been
produced from embryonic stem cells (ESCs) by recapitulating embryonic development in
vitro. However, problems associated with the use of ESCs mean that an alternative cell
source is needed. In 2006, it was discovered that 4 transcription factors can reprogram
somatic cells into induced pluripotent stem cells (iPSCs). iPSCs provide an alternative source
of pluripotent stem cells and can be derived in a patient-specific manner. iPSCs have been
shown to differentiate in vitro into insulin-expressing cells, but it is unknown whether iPSCs
are truly equivalent to ESCs. Important differences have been shown to exist between iPSCs
and ESCs which may affect the ability of iPSCs to give rise to cells of a pancreatic lineage
and therefore limit their usefulness for the treatment of T1DM. The aim of this project is to
identify whether iPSCs are a viable alternative to ESCs for generating β-cells in vitro for cell
replacement therapy to treat type 1 diabetes. The differentiation potential of iPSCs and ESCs
to give rise to first definitive endoderm (the first stage in differentiation towards a pancreatic
lineage) in vitro will be compared, and the involvement of miRNAs in differentiation of
ESCs and iPSCs to definitive endoderm will be investigated.
Table Of Contents
Chapter 1. General Introduction...........................................................................................4
1.1 Diabetes Mellitus..............................................................................................................8
1.1.1 Type 1 Diabetes Mellitus (T1DM) .........................................................................8
1.1.2 Type 2 Diabetes Mellitus (T2DM) .........................................................................9
1.1.3 Other Types of Diabetes .......................................................................................10
1.2 Structure of the Pancreas ................................................................................................11
1.3 Developmental Biology of the Pancreas ........................................................................12
1.3.1 Formation of Definitive Endoderm....................................................................12
1.3.2 Pancreatic Specification .....................................................................................14
1.3.3 Human Pancreatic Development .......................................................................22
1.4 Insulin and Its Mechanisms of Action............................................................................23
1.4.1 Insulin Biosynthesis ............................................................................................23
1.4.2 Insulin Secretion..................................................................................................24
1.4.3 Insulin Action ......................................................................................................25
1.5 Treatment of Type 1 Diabetes ........................................................................................27
1.5.1 Insulin Replacement ..........................................................................................27
1.5.2 Pancreas Transplantation ...................................................................................29
1.5.3 Islet Transplantation ..........................................................................................29
1.5.4 Xenotransplantation ...........................................................................................31
1.6 Cell Replacement Therapy .............................................................................................33
1.6.1 Human β-cells .....................................................................................................33
1.6.2 Pancreatic Stem Cells ..........................................................................................35
1.6.3 Other Adult Stem Cells .......................................................................................38
1.7 Embryonic Stem Cells (ESCs) .......................................................................................43
1.7.1 Differentiation of ESCs into Pancreatic β-cells ....................................................45
1.8 Induced Pluripotent Stem Cells ......................................................................................55
1.8.1 Differentiation of iPSCs into Pancreatic β-cells ....................................................57
1.8.2 Considerations Prior to Clinical Application of iPSCs .........................................61
1.9 Differences Between iPSCs and ESCs ...........................................................................70
1.9.1 Gene Expression ...................................................................................................71
1.9.2 Genomic Stability.................................................................................................74
1.9.3 Epigenomic Stability ............................................................................................77
1.9.4 Epigenetic Memory ..............................................................................................78
1.9.5 miRNA Expression ..............................................................................................81
1.10 MicroRNA ....................................................................................................................82
1.10.1 Biosynthesis and Action of miRNAs ................................................................83
1.10.2 The Role of miRNAs in Pluripotency ...............................................................84
1.10.3 The Role of miRNAs in Differentiation............................................................88
1.11 Hypothesis & Aims ......................................................................................................95
Chapter 2: Materials & Methods…………………………………………………………..97
2.1 Generation of Induced Pluripotent Stem Cell (iPSC) Lines ........................................100
2.1.1 Preparation of Cell Lines ..................................................................................100
2.1.2 Preparation of pMX Plasmids ...........................................................................102
2.1.3 Reprogramming of Fibroblasts .........................................................................104
2.2. Maintenance of Pluripotent Stem Cells in Culture......................................................107
2.2.1 SNL Feeder Cell Culture & Inactivation ..........................................................107
2.2.2 Maintenance of Pluripotent Stem Cells on Feeder Cells ..................................109
2.2.3 Preparation of Matrigel™-Coated Plates ..........................................................110
2.2.4 Maintenance of Pluripotent Stem Cells on Matrigel™ .....................................111
2.2.5 Cryopreservation of Stem Cells ........................................................................111
2.3. In Vitro Differentiation of Stem Cells.........................................................................113
2.3.1 Differentiation to Definitive Endoderm ............................................................113
2.4. Characterisation of iPS Cells.......................................................................................114
2.4.1 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) ........................114
2.4.2 Gel Electrophoresis ...........................................................................................118
2.4.3 Quantitative PCR (qRT-PCR) for Detection of mRNA Expression .................119
2.4.4 Immunocytochemistry.......................................................................................122
2.5 Investigation of microRNA Expression .......................................................................124
2.5.1 Microarray Analysis ..........................................................................................124
2.5.2 qRT-PCR for Detection of miRNA Expression ................................................129
2.5.3 Identification of Gene Targets of miRNAs .......................................................132
2.5.4 Luciferase Assay in 293FT Cells ......................................................................135
Chapter 3: Generation and characterisation of induced pluripotent stem cell lines.....141
3.1 Introduction ..................................................................................................................143
3.2 Methods ........................................................................................................................145
3.2.1 Generation of induced pluripotent stem cells....................................................145
3.2.2 Maintenance of pluripotent stem cells in culture ..............................................146
3.2.2 Characterisation of pluripotent stem cells .........................................................146
3.3 Results ..........................................................................................................................149
3.3.1 Generation of New iPS Cell Lines ....................................................................149
3.3.2 Characterisation of New iPSC Lines ................................................................152
3.4 Discussion ....................................................................................................................163
Chapter 4: Differentiation of pluripotent stem cells into definitive endoderm.………170
4.1 Introduction ..................................................................................................................171
4.2 Methods ........................................................................................................................173
4.2.1 Differentiation of Pluripotent Stem Cells into Definitive Endoderm .......................173
4.2.2. Characterisation of Differentiated Cells ...................................................................174
4.3 Results ..........................................................................................................................176
4.3.1 Comparison of Differentiation Protocols ..........................................................176
4.3.2 Comparison of Cell Lines .................................................................................181
4.4 Discussion ....................................................................................................................186
Chapter 5: Identification of miRNAs that play a role in the formation of definitive
endoderm..............................................................................................................................194
5.1 Introduction ..................................................................................................................197
5.1.1 The role of miRNAs in Pluripotency and Differentiation .................................197
5.1.2 The Role of miRNAs in Definitive Endoderm Formation ................................200
5.1.3 Differences in miRNA Expression between ESCs and iPSCs ..........................200
Aims ...................................................................................................................................202
5.2 Methods ........................................................................................................................203
5.2.1. Choice of Cell Lines.........................................................................................203
5.2.2 In Vitro Differentiation of Pluripotent Stem Cells into Definitive Endoderm..203
5.2.3 Microarray Analysis ..........................................................................................203
5.2.4 qRT-PCR For miRNA Expression ....................................................................204
5.3 Results ..........................................................................................................................205
5.3.1 Analysis of Microarray Results .........................................................................205
5.3.2 Validation of Microarray Results by qRT-PCR ................................................222
5.4 Discussion ....................................................................................................................239
Chapter 6: Investigation of the function of miRNAs in differentiation to definitive
endoderm……...............................................................................................................242
6.1 Introduction ..................................................................................................................247
6.2 Methods ........................................................................................................................249
6.2.1 Identification of Gene Targets of miRNAs .......................................................249
6.2.2 Luciferase Assay in 293FT Cells ......................................................................249
6.2.3 Manipulation of miRNA Expression ................................................................250
6.3 Results ..........................................................................................................................251
6.3.1 Identification of Gene Targets of miRNAs .......................................................251
6.3.2 Investigation of the Function of miR-375 in Definitive Endoderm Formation 251
6.3.3 Investigation of the Function of miR-151a-5p in Definitive Endoderm
Formation ...................................................................................................................258
6.3.4 Manipulation of miRNA Expression in Pluripotent Stem Cells .......................262
6.4 Discussion ....................................................................................................................267
Chapter 7: General discussion .....................................................................................269
7.1 Generation and Characterisation of iPSCs ...................................................................273
7.2 Differentiation of Pluripotent Stem Cells into Definitive Endoderm ..........................275
7.3 Investigation of miRNA Expression in DE Formation ................................................278
7.4 Investigation of Differential miRNA Expression between ESCs and iPSCs ...............280
7.5 Investigation of the Function of miRNAs in DE Formation ........................................283
7.6 Concluding Remarks ....................................................................................................287
Bibliography……………………………..…………………….……….……………....…...290
Appendix 1: Supplementary miRNA tables………………………………....….…........…334
Appendix 2: miRNA target predictions………………………………..………………......344
Appendix 3: List of Materials…………………………………………...…………............350
List of Figures
Figure 1.1. Structure of the pancreas ............................................................................. 11
Figure 1.2. Early development of the dorsal pancreas. .................................................. 14
Figure 1.3.Endocrine specification in the developing pancreas. .................................... 19
Figure 1.4. Approximate timescale of mouse and human pancreatic development ........ 22
Figure 1.5. Signal transduction in β-cells ...................................................................... 24
Figure 1.6. The insulin signalling pathway ................................................................... 26
Figure 1.7. Derivation of ESC lines from cleavage-stage embryos................................ 43
Figure 1.8. Differentiation of pluripotent stem cells toward insulin-producing β-cells in
vitro recapitulates in vivo development ......................................................................... 48
Figure 1.9. Derivation of iPSCs & their applications in cell replacement therapy, disease
modelling and gene therapy. .......................................................................................... 56
Figure 1.10 miRNA biogenesis ..................................................................................... 84
Figure 1.11. miRNAs expressed at the different stages of pancreatic development ....... 91
Figure 2.1. Overview of the process of reprogramming of somatic cells to induced
pluripotent stem cells. .................................................................................................. 106
Figure 2.2. Passage of stem cell colonies.. .................................................................. 109
Figure 2.3. Amplification plot. .................................................................................... 121
Figure 2.4. Standard curve .......................................................................................... 121
Figure 2.5. Overview of microarray analysis of microRNA expression....................... 126
Figure 2.6. Typical electropherogram from an RNA 6000 Nano Bioanalyzer chip used to
analyse RNA quantity and quality. .............................................................................. 127
Figure 2.7. Normalisation of data using Lowess normalisation. .................................. 128
Figure 2.8. Plasmid used in luciferase assays to determine miRNA-target relationships..
.................................................................................................................................... 137
Figure 2.9 miArrest™ lentiviral vector used to repress expression of miRNAs of
interest. ........................................................................................................................ 137
Figure 2.10 miExpress™ lentiviral vector used to overexpress miRNAs of interest. ... 137
Figure 2.11. Overview of luciferase assay to detect miRNA-target interactions. ......... 139
Figure 3.1 GFP expression during reprogramming ...................................................... 150
Figure 3.2. Formation of stem cell-like colonies after transfection of HFF1 fibroblasts
with reprogramming factors ......................................................................................... 150
Figure 3.3. Immunocytochemistry for TRA-1-60 expression using live cell stain. ...... 152
Figure 3.4. qRT-PCR showing expression levels of key pluripotency genes. .............. 153
Figure 3.5 Morphology of stem cell colonies. ............................................................. 154
Figure 3.6. Immunocytochemistry for TRA-1-60 expression. ..................................... 155
Figure 3.7. Alkaline phosphatase staining...................................................................155
Figure 3.8. Expression of pluripotency markers. ......................................................... 156
Figure 3.9. Expression of pluripotency markers. ......................................................... 156
Figure 3.10. Expression of pluripotency markers. ....................................................... 157
Figure 3.11 Expression of pluripotency markers ......................................................... 157
Figure 3.12. qRT-PCR showing expression levels of key pluripotency genes ............. 159
Figure 3.13. qRT-PCR showing expression levels of exogenous genes used in
reprogramming. ........................................................................................................... 159
Figure 3.14. qRT-PCR data showing expression of marker genes characteristic of
endoderm. .................................................................................................................... 161
Figure 3.15. qRT-PCR data showing expression of a marker gene characteristic of
mesoderm .................................................................................................................... 161
Figure 3.16. qRT-PCR data showing expression of marker genes characteristic of
ectoderm. ..................................................................................................................... 161
Figure 3.17 qRT-PCR data showing the expression of the pluripotency genes OCT4 and
NANOG in two iPSC lines at several different passages. ............................................ 165
Figure 4.1.Immunocytochemistry for SOX17 expression. ........................................... 176
Figure 4.2.Expression of Sox17 assessed by qRT-PCR ............................................... 178
Figure 4.3. Expression of Cxcr4 assessed by qRT-PCR .............................................. 178