Download Germ lineage specification from a pluripotent primitive ectoderm

Germ lineage specification from a pluripotent
primitive ectoderm-like substrate: a role for cell-cell
contacts
A thesis submitted to the University of Adelaide for the degree of Doctor of Philosophy
By
James Nicholas Hughes, B.Sc. (Hons)
Department of Biochemistry
School of Molecular and Biomedical Sciences
The University of Adelaide
Adelaide, South Australia
March 2008
Summary
Statement
Acknowledgements
iv
vi
vii
Chapter 1: Introduction
1
1.1 The promise of ES cells
1
1.2 In vitro differentiation: embryoid bodies (EB)
2
1.3 Lessons from the embryo and equivalence to in vitro systems
3
1.3.1 Primitive Ectoderm formation in the mouse
3
1.3.2 Primitive ectoderm formation in vitro
6
1.3.2.1 MEDII
7
1.3.2.2 EpiSCs
8
1.3.3 From primitive ectoderm to committed germ lineages in the embryo
8
1.3.4 Establishing the signalling environment for lineage specification
10
1.3.4.1 Proximal-distal Axis
10
1.3.4.2 Anterior-posterior Axis
12
1.3.5 Mesendoderm specification in the posterior primitive ectoderm
13
1.3.5.1 BMP
13
1.3.5.2 WNT
14
1.3.5.3 Nodal
15
1.3.5.4 FGF
15
1.3.5.5 E-cadherin
16
1.4 Mesendoderm induction from ES cells
17
1.5 Differentiation of mesoderm and endoderm derivatives from ES cells
20
1.6 Ectoderm formation: A role for active mesendoderm suppression in vivo and in vitro
22
1.7 ES cell differentiation can be controlled by the physical environment
23
1.8 EPL cells represent a superior starting population to ES cells for investigating lineage commitment
24
1.8.1 Ectoderm from EPL cells
25
1.8.2 Mesendoderm from EPL cells
26
1.9 Anterior/posterior model
27
1.10 Thesis Aims
29
Chapter 2: Materials and Methods
31
2.1 Nomenclature
2.2 Abbreviations
2.3 Cell culture
2.3.1 ES Cell Lines
2.3.2 Media components and additives
2.3.3 Media
2.3.4 Feeder free ES cell maintenance
2.3.6 Feeder dependent ES cell maintenance
2.3.6.1 MEF isolation and storage
2.3.7 MEF depletion
2.3.8 Differentiation of ES cells to adherent EPL cells
2.3.9 Differentiation of ES cells to suspension EPL cells
2.3.10 EPLEBs from adherent EPL cells
2.3.11 EPLEBM and EPLEB from suspension EPL cells
2.3.12 EPLIC
2.3.13 EBM
2.4 Differentiation Assays
2.5 Primers
31
31
32
32
33
34
34
34
35
35
35
36
36
36
37
37
37
37
2.6 Antibodies
2.7 RNA isolation
2.8 cDNA synthesis
2.9 Tissue collection from mice
2.10 DNA isolation
2.11 Northern blot analysis
2.11.1 Synthesis of radiolabelled DNA probes
2.11.2 Hybridisation of 32P-labelled probes to Northern blots
2.12 qPCR analysis
2.13 Cellular Protein Extraction
2.13.1 SDS-PAGE and Western blot analysis
2.14 Transient transfection
2.15 Immunofluorescence
2.16 Paraffin embedding, microtome sectioning H&E staining
2.16.1 Hematoxylin and Eosin (H&E) staining
2.17 Animal Work
2.18 Preparation of single cells suspensions
2.18.1 Flow Cytometry
41
41
41
41
42
42
42
43
43
43
44
44
45
45
45
46
46
46
Chapter 3: Defining EPL cell production from ES cells and comparison of mesoderm differentiation in
Embryoid Bodies derived from ES and EPL cells
47
3.1 Introduction
47
3.2 ES cells grown for 1, 2, 3 or 4 days in MEDII either adherently or in suspension all display EPL cell
gene expression
48
3.3 Kinetics of EPL cell differentiation to mesoderm depends on the length of culture in MEDII
49
3.4 ES cells begin transition to EPL cells within 24 hours of MEDII exposure in suspension culture 54
3.5 Defining the EPL cell stage
56
3.6 EPLEB model primitive streak progression and share key EMT markers with the embryo
61
3.7 EPLEB differentiate to primitive blood faster than ESEB
64
3.8 Discussion
68
3.8.1 Equivalence of suspension and adherent culture for EPL cell formation
68
3.8.2 EPL cells model posterior differentiation when differentiated as EPLEB
68
3.8.3 Blood formation in EPLEBs resembles YS hematopoiesis
69
3.8.4 A transitional stage between ES and EPL cells exists after 24 hours MEDII exposure 69
3.8.5 Dynamic acquisition of EPL cell identity and equivalence to primitive ectoderm formation
in the embryo
70
3.8.6 Are functional changes associated with maturing EPL cells?
70
Chapter 4: Neither EPL nor ES cells nor their early derivatives contain detectable LTR-HSC activity 72
4.1 Introduction
4.2 Establishment of radio-protection assay
4.3 R1-GFP ES cells undergo transgene silencing in a proportion of differentiated progeny
4.4 Assessment of EPL and ES derived cell populations for short-term radioprotection
4.5 Neither EPL nor ES cells provide short-term radio-protection
4.6 Discussion
72
74
74
76
82
82
Chapter 5: Controlling mesoderm formation from EPL cells
85
5.1 Introduction
85
5.2 Both Trypsinisation and MEDII withdrawal are important in mesoderm differentiation from EPL cells
87
5.3 Trypsinisation of EPL cells favours mesoderm formation at the expense of ectoderm by an unknown
mechanism
87
5.3.1 Differentiation outcome in response to trypsinisation
87
5.3.2 Morphology correlates with differentiation outcomes
92
5.3.3 Trypsinisation induces gene expression changes consistent with alterations to
differentiation outcome
92
5.4 Disaggregation using multiple agents is equally effective in suppressing ectoderm from EPL cells96
5.5 MEDII favours ectoderm formation from EPL cells by an unknown mechanism
98
5.6 MEDII window of action
102
5.7 Matrigel does not favour ectoderm differentiation from EPL cells
104
5.8 Discussion
104
5.8.1 Disaggregation appears to sensitise pluripotent cells to mesoderm inducing signals 104
5.8.2 MEDII appears to substitute for AVE signalling
106
5.8.3 EPL cells retain plasticity after disaggregation
106
Chapter 6: Staurosporine induces mesoderm from EPL cells via an EMT
109
6.1 Introduction
109
6.2 EPL colonies display morphological EMT in response to SSP
110
6.3 25nM SSP favours mesoderm differentiation at the expense of ectoderm differentiation from EPL
cells
113
6.4 Length of SSP exposure has an effect on differentiation outcome
115
6.5 EPL cells exposed to SSP for 24 hours in the presence of MEDII take on a mesodermal gene profile
117
6.6 Discussion
119
6.6.1 SSP induces morphological changes but not full mesenchymal identity in the presence of
MEDII
119
6.6.2 SSP alters the differentiation outcome in favour of mesoderm
121
6.6.3 E-cadherin transcript levels drop before up-regulation of Snail1
121
6.6.4 Cells remain responsive to MEDII after SSP treatment
122
Chapter 7: γ-secretase inhibition favours ectoderm differentiation from EPL cells
123
7.1 Introduction
7.2 γ-secretase inhibition favours ectoderm formation at the expense of mesoderm from EPL cells
7.3 γ-secretase inhibition has no effect on the formation of EPL cells from ES cells
7.4 Active Notch1 signalling is not detectable in EBM or EPLEB
7.5 Stimulation of EPL cells with Notch ligand does not alter differentiation outcome
7.6 Ectoderm induction by γ-secretase inhibition is mediated by E-cadherin stabilisation
7.7 Discussion
7.7.1 γ-secretase is active in mesoderm specification from EPL cells
7.7.2 γ-secretase might operate by blocking E-cadherin proteolysis
7.7.3 Inhibition of γ-secretase does not inhibit EPL cell formation but appears to block
differentiation prior to mesoderm specification, at a similar stage to MEDII
7.7.4 Notch signalling is not required for neurectoderm formation
123
124
133
135
138
140
146
146
147
148
149
Chapter 8: Control of mesoderm formation from primitive ectoderm by disruption of the epithelial cell
state
151
8.1 Understanding and controlling early events in lineage determination will be important for delivering
desired cell types from pluripotent cells
151
8.2 Integration of MEDII, cell dissociation and serum in controlling EPL cell differentiation
151
8.3 EPL cells respond to modulation of epithelial state by differentiating to alternate outcomes
152
8.4 ES and EPL cells respond differently to disaggregation
154
8.5 The effect of disaggregation is likely to involve γ-secretase independent mechanisms
154
8.6 Disruption of epithelial state can directly initiate EMT prior to Snail1 up-regulation
156
8.7 EBM represent a good model of anterior morphogenesis
158
8.8 MEDII suppression of Mesoderm in the presence of serum relies on the maintenance of cell:cell
contacts
158
8.9 Relevance to hES cells
158
8.10 Final remarks
160
References
161
Summary
During mammalian development a small number of pluripotent cells proliferate and differentiate to give
rise to all the mature cell types of the organism. Among the earliest differentiation events is the process
of gastrulation, in which pluripotent primitive ectoderm cells form the three germ lineages, mesoderm,
ectoderm and endoderm under the control of complex signalling and environmental cues. This process
can be modelled using embryonic stem cells, which have proven to respond to embryologically relevant
signals during in vitro differentiation and promise to uncover additional insights into the process of germ
lineage specification. This thesis describes the differentiation of mouse ES cells to committed cell types
via a second intermediate population of pluripotent cells termed Early Primitive Ectoderm-Like (EPL)
cells. The similarity of EPL cells to primitive ectoderm and the rapid acquisition of lineage specific
markers and loss of pluripotent characteristics upon differentiation of EPL cells suggest they are an
excellent model for the cells in the embryo that undergo germ lineage commitment.
EPL cells can be differentiated as EPLEBs, which are highly enriched in mesodermal cell types and
contain essentially no ectodermal derivatives and no visceral endoderm. Here it is shown that EPLEBs
can be generated from EPL cells grown either adherently or in suspension culture provided the cells are
reduced to a single cell suspension before reaggregation as EPLEBs. Since EPLEBs are a rich source
of mesoderm and contain less non-mesodermal cell types than traditional ESEBs, they were assayed
for definitive blood formation, however none was detected.
Alternately, EPL cells can be differentiated in the presence of MEDII in aggregates termed EBMs, which
are restricted to ectodermal cell fates. Here it is demonstrated that the switch from mesodermal to
ectodermal differentiation observed in ELPEBs and EBMs relies on two variables; a mesoderm
suppressing activity within MEDII and the pro-mesodermal activity of cell dissociation as undertaken
during EPLEB formation.
Evidence has been presented that interventions that modulate the epithelial identity of EPL cells are
capable of influencing subsequent differentiation such that protection of the epithelial cell state favours
ectoderm while disruption favours mesoderm. Staurosporine (SSP) is a kinase inhibitor that has been
shown to induce an epithelial to mesenchymal transition in chick neural tube. Here it was added to EPL
cells with the result that mesodermal differentiation was enhanced at the expense of ectoderm. DAPT is
a potent inhibitor of γ-secretase, which cleaves a number of protein targets including the adherens
junction component E-cadherin. Addition of DAPT to differentiating EPL cells has the opposite effect to
SSP, with an increase in ectodermal differentiation at the expense of medoderm. It is proposed that
DAPT is acting by preventing E-cadherin cleavage and thus stabilising the epithelial state.
Modulation of epithelial contacts between pluripotent cells represents a novel way to control lineage
induction and as such the incorporation of these findings into methodologies for directed differentiation
in defined culture conditions is likely to provide improved outcomes in the production of desired cell
types.
Statement
This work contains no material that has been accepted for the award of any other degree or diploma in
any university or other tertiary institution and, to the best of my knowledge and belief, contains no
material previously published or written by another person, except where due reference has been made
in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being made available
for loan and photocopying, subject to the provisions of the Copyright Act 1968.
SIGNED…………………………….
DATE……………………………….
Acknowledgments
I would like to thank Grant Booker for the opportunity to undertake a Ph.D. in the Department of
Biochemistry.
My thanks also go to Peter Rathjen for accepting me as a student within his laboratory. To Joy Rathjen,
who has done more than any other to ensure the success of this work, I am truly grateful for your
support and insights into both the science of embryonic stem cells and the process of completing a
Ph.D.. I would especially like to thank you for the many hours you spent reading this manuscript and
pointing out the innumerable occasions where I had failed to adequately expound my arguments. To
Mike Morris and Rebecca Keough, thanks for all your input into my project and for your part in my
scientific development.
A Ph.D. would be an awfully long road if you didn’t enjoy each day and it was because of all the people
in the lab and the department as a whole that I can honestly say I have thoroughly enjoyed the last four
years. Quite a few people have come and gone over that time but the main characters who shared a
laugh with me were, my PhD classmates, Harves, Tiffany, Sarah, Phil and Ana, the lab clowns, Mike
and Kenneth and my good mates, Lynda, Fernando, Brett, Svetlana, Colleen, Ljiljana, Rob, Tetyana
and Nori. Thanks to John Mac, who was an enormous help with all things TC and to Jan for fixing all my
dodgy orders.
As with all major undertakings in life it’s only worth it if you’re got the support of the people you care
about and that was certainly the case with my PhD. To Mum and Dad and all my family I extend my
genuine thanks for your love and encouragement. The last person I’d like to thank is the person to
whom I am most indebted, my wife Beck, without whose support and encouragement I might never
have finished. She put up with an absent husband for so many weekends and even managed to sound
interested when I’d come home from the lab rabbiting on about some new result. It’s because of Beck
that I count the last four years of my life as such a success and now with our new son Oscar I look
forward to the next exciting chapter of our lives together.