The relationship between pluripotency state and susceptibility of nuclear shape and organisation to cell tractionin mouse ESCs

Oliver Brookes1,2, Stephen D Thorpe1, Julien E Gautrot1,2, David A Lee1

1School of Engineering and Material Science and2Institute of Bioengineering, Queen Mary University of London

Embryonic stem cells lack the thickened lamina and perinuclear actin that regulates nuclear shape in adult stem cells and terminally differentiated cells. It is not clear whether this heightened mechanical susceptibility is incidental or whether it is an evolved feature that fulfils a specific role in pluripotency or differentiation.The early embryo, in which pluripotent cells are found in vivo, is not subject to external mechanical loading. Therefore, we reason that cell-generated forces are the only native mechanical challenge encountered by embryonic stem cells. We hypothesise that distortion of ESCs by intracellular forcescan induce changes in nuclear organisation that influence maintenance of pluripotency or induction of differentiation.

Using micropatterned substrates, we aim to create anisotropic regimes in which cytogenic forcescause cellular and nuclear elongation. ESCs growing on surfaces treated with gelatin (an established substrate for ESC culture) exhibit relatively poor adhesion, so we utilise different modes of specific adhesion (cell-cell or cell-matrix) to prompt a morphological response. Cells cultured on either fibronectin or E-cadherin adopt comparable morphologies, both being flatter and more spread than cells grown on a minimally adhesive gelatin substrate (see poster for figure). Fibronectin is known to induce differentiation and impacts expression of pluripotency markers, introducing this as another variable for study. Confocal and live imaging of cells stained with H33342 is utilised to investigate nuclear shape,shape stability and chromatin organisation which may in turn relate to differences in function on a genetic or epigenomic level.

Extended Abstract

The early embryo is not subject to external mechanical loading. Therefore, we reason that cell-generated forces are the only native mechanical challenge encountered by embryonic stem cells. Here we investigate whether these forces can create changes in nuclear organisation that might influence maintenance of pluripotency or induction of differentiation.

We describe culture of cells in geometrically constrained conditions, specifically confining cell adhesion to narrow lanes with a surface-bound polymer brushwhich prevents protein adsorption and therefore cell adhesion outside the lanes (Fig 1A). Intracellular forces are variably able to induce cellular and nuclear elongation, the extent of distortion being dependent on adhesion mode. Cells cultured on fibronectin or E-cadherin coated surfaces adopt a significantly flatter and more spread morphology than cells grown on a gelatin-coated substrate (Fig 1C). The extent of this change is also related to pluripotent state, lower expression of REX1 or SSEA1 being characteristic of more spread cells (Fig 1B).Live and confocal imaging of cells stained with H33342 reveals differences in nuclear shape, stability and chromatin organisation which may in turn relate to differences in function on a genetic or epigenomic level. Preliminary results suggest that nuclear volume may be influenced by nuclear elongation (Fig 1E), possibly because lateral confinement reduces flattening of the cell and therefore reduces force on the nucleus.Furthermore, susceptibility of nuclear shape to cellular distortion appears to increase as pluripotency marker expression is reduced, but nuclear stability seems to diminish.

Figure 1.The microprinting (A,i-iii) and polymer brush growth process (A,iv-v). Cells grow in the bare lanes and not on the polymer brush. Growing cells on unpatterned fibronectin surfaces induces a larger spread area, but may suppress expression of pluripotency associated genes (B). ESCs grown on E-cadherin coated surfaces adopt a flatter, individualised growth habit compared to the same population grown on gelatin (C, non-patterned 2i cells shown). Similar morphology is seen for cells grown on fibronectin (not shown). Cells grown on fibronectin-coated lanes adopt an elongated morphology aligned with the lanes, as do their nuclei (D). Preliminary results suggest that culture on these micropatterns may induce changes in nuclear volume (E).

Oliver Brookes

Curriculum Vitae

I am an experienced researcher, interested in stem cell research, regenerative medicine and tissue engineering.

Email: ,

Tel: +44 (0) 7512 073393

Born: 22/04/1982, Bath, UK (British nationality)

Current occupation:

I am nearing completion of a PhD on the role of nuclear mechanotransduction in pluripotency and differentiation of mouse ESCs. The experimental approach here is based on the use of micropatterned two- and three-dimensional culture substrates (which I manufacture myself) to confine cells to specific morphologies. The cytoskeletal and nuclear architecture of the cells is then interrogated using epifluorescent, confocal and super-resolution microscopy. Analysis and interpretation of the images produced is conducted with the use of several automated computational routines, some of which I programmed personally. As such I can lay claim to skills and knowledge in microfabrication, polymer chemistry, cell culture, microscopy, programming and statistical analysis.

Academic Qualifications:

2011 – 2012

University of Sheffield

MSc Stem Cell and Regenerative Medicine: Graduated with merit

Half of the marks for this accreditation come from taught courses, and half from a research project. Taught content has comprehensively covered topics relevant to stem cells and their niches, as well as interdisciplinary subjects like bionanomaterials, and supporting subjects like research ethics.

My research project (under the supervision of GuiseppeBattaglia) concerned the development of an in vitro model of the murine blood-brain barrier based on immortalised cell lines. Mouse endothelial cells in culture do not readily form a stringent barrier like that seen in vivo. In the course of my project, I developed atheoretical understanding of this based on my experiments, and went on to test uptake of fluorescent polymersomes. Polymersomes are supramolecular structures similar to liposomes, and were being investigated as a potential drug-delivery platform. Therefore, my project consisted not only of the development of an in vitro neuroendothelial system, but also in its use as a physiological model for drug delivery.

2000 – 2003

University of Wales, Aberystwyth

BSc Biochemistry and Biotechnology 2:1 (hons)

This degree included a research project, which focussed on the creation and affinity purification of a novel calcium-sensitive YFP-GST fusion protein via cloning into a commercial plasmid which was then electroporated into bacterial cells.

In the course of the degree I also voluntarily attended an additional biochemistry module, not included on the transcript.

1998 – 2000

John of Gaunt School

A level Biology, Chemistry and Geology (B, C, B respectively)

1993 – 1998 Lavington Comprehensive School

8 GCSEs including English and Mathematics

Professional experience

2007 – 2010

Helmhotz-ZentrumMünchen, Germany.

Marie Curie Early Stage Researcher

I was employed in the Institute of Experimental Genetics at Helmholtz-zentrumMuenchen, a research institute in Munich, Germany. The project I worked on here concerned the controlled differentiation of several somatic cell types from murine ESCs, and reprogramming of differentiated cells by microinjection of ESC nuclear extracts. In addition to the routine culture of mESCs, and workhorse techniques such as PCR and immunofluorescent microscopy, I also used some advanced methods such as spectral karyotyping (SKY) and microinjection into cell nuclei.

This position was part of a Marie Curie research training network. As a member of the Clonet network, I received training in professional skills (ethics, communication, spin-out business and intellectual property) in addition to a wide program of training in scientific methods such as advanced microscopy, in-situ hybridisation and microarray analysis.

2004 – 2007

University of Nottingham, UK.

Research Technician and PhD student (Part-time, Withdrawn)

After graduating, I worked and studied at the University of Nottingham, in the group of the late Keith Campbell. Here I worked on several projects concerning the derivation of pluripotent cells for domestic livestock species. One of these projects, on using the controlled expression of early embryonic genes to establish and maintain ESCs from porcine blastocysts, was intended to be my PhD project. However, no funding could be found, and despite my having worked on the project for some time as a self-funded student, it eventually had to be abandoned.

During this time I routinely used in vitro embryological methods to create blastocysts, molecular biological and proteomic methods to isolate genomic DNA, mRNA and proteins, and analytical methods such as immunofluorescent microscopy, SDS-PAGE and PCR. I also worked as a research technician on other projects, including the isolation and primary culture of stem cells from somatic tissues including bone marrow, muscle and adipose tissue.

Other experience

2010 – 2011 and 2012 – current

University of Bath - Student support worker.

I provide academic support for students with disabilities, taking notes for them in lectures or assisting in practical classes. The content I have covered while working in this capacity is more than equivalent to a second degree, and has covered many topics in chemistry and pharmacy, as well as aspects of biology that were not part of my degree.

2010 – Conservation volunteering

During this period I operated as a conservation volunteer in Fiji and Australia, acquiring several non-academic qualifications along the way, including substantial experience in conducting surveys and transects to collect data on biodiversity and species populations.

2003 – 2004 - Travelling in Australia

Personal Qualities:

  • I am outgoing and articulate – I enjoy meeting and interacting with people, enjoy working with other people to achieve common goals, and I am not afraid of public speaking or debate.
  • I enjoy working and socialising with international colleagues and have a foundation in several languages beside my native English.
  • I am compassionate and enjoy helping others. I like sharing skills and knowledge with friends and colleagues, and have been told that I am a patient teacher.
  • I consider myself a fast learner, able to adapt to changing conditions and new ways of working, and I enjoy intellectual challenges and opportunities to learn.