Stem cell organisation and differentiation in the niche

Lead Research Organisation: University College London
Department Name: Division of Pathology

Abstract

There is an expectation that stem cells will one day be able to cure a wide variety of human diseases. However, development of stem cell therapies relies upon a sound understanding of the biology of these fascinating cells. Stem cells occupy a specialised environment in the body known as a ?niche? which controls stem cell behaviour and survival. Most human stem cell niches are deep in the body and so difficult to study. The outermost layer of the cornea on the front surface of the eye has stem cells which live in a transparent area called the limbus. We have recently discovered new structures in the limbus which we believe may be the stem cell niche. We propose to study these structures and the cells associated them in donated human corneas using techniques including scanning electron and confocal microscopy determine where exactly the stem cells are and how they function. We will also study the effect of age upon the stem cells and their niche. This is very important because clinically it is possible to take a small biopsy of the healthy limbus to prepare stem cell therapy for patients blinded by specific injuries or diseases of the outermost layer of the cornea. However, we and others are providing this therapy without knowing the optimal biopsy location. Our study will therefore improve this surgical technique. We must also be able to refine treatments for individual patients which requires knowledge of what happens to the stem cells after they have been transplanted. We propose to develop a technique for labelling and transplanting stem cells onto the human cornea using very small particles which fluoresce brightly and can be detected with clinical instruments. Using this methodology we will also determine how well stem cells organise themselves in the niche after transplantation and if they have any affect upon that niche. Our data will help us to understand one of the important features of the eye which contributes to clear vision (i.e. healthy, functioning stem cells in the limbus) which will help to improve stem cell therapy for the cornea. If it is found that our data provide parallels with other human stem cell niches, it would support the use of the readily accessible and transparent cornea as a model for the development of other stem cell therapies.

Technical Summary

This research proposal reflects the current scientific and clinical focus on stem cells in health and disease. Occupation of a specialised niche environment is important for stem cell survival and function; however, in the main these niches are difficult to study in humans. In the transparent cornea, a population of limbal epithelial stem cells (LESCs) constantly regenerates the epithelium providing an excellent model for studying an adult stem cell niche. LESC deficiency is a serious blinding condition with no accepted cure. Cultured LESC therapy is being trialled clinically in laboratories around the world including ours. However, the characteristics of this stem population and its niche (the limbus) are poorly understood. Two of the key elements of LESC therapy efficacy are the quality of the biopsy from which LESC are originally harvested for in vitro expansion and the condition of the recipient host environment, both of which are have been hampered by a lack of technology to clearly visualise the niche and transplanted cells over time. We have recently identified novel structures (crypts and focal stromal projections) within the limbus which we propose to be a putative LESC niche owing to the characteristics of the resident cells. We propose to build upon these findings to further test our hypothesis of the existence of heterogeneity in the LESC niche and also determine the effect of age upon the cellular and architectural components. These studies have particular clinical relevance as they will provide data to direct the location from which a limbal biopsy should ideally be taken for LESC therapy production. Currently biopsy harvest location is not based on experimental evidence. Furthermore we will evaluate the practicality of using fluorescent semi-conductor nanocrystals (QDots) for labelling and tracking of transplanted cells on the human cornea using an ex-vivo culture system ? a technology with future clinical potential. Specifically we will assess the ability of the de-cellularised LESC niche to support the re-organisation of cultured limbal epithelial cells, including LESC, to their locations in normal tissue. The ?potency? of the niche for directing stem cell fate will be tested using GFP labelled mouse embryonic stem cells. Finally, we will assess the regenerative capacity of the LESC niche in our model systems. This is an ambitious element of our work but one which may shed light on the mechanisms of LESC therapy efficacy and failure which are currently not understood.
 
Description First in human phase I / II clinical trial of RAFT for aniridia related keratopathy.
Amount £2,892,577 (GBP)
Funding ID MR/S018883/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 07/2023
 
Description TSB - Cell Therapy Autumn 2007 TS/G000611/1 Rapid Automated Fabrication of Tissues (RAFT): Corneal Stem Cells
Amount £753,068 (GBP)
Funding ID TS/G000611/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 06/2016
 
Title Human corneal epithelial (spontaneous) cell line - HCE-S 
Description We developed a spontaneously immortalised human corneal epithelial cell line with retained characteristics of stem cells. This cell line has been requested and passed on to other researchers. 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact The experiments are ongoing. 
 
Title In vitro pig model of limbal stem cell deficiency, transplantation and cell tracking 
Description A 3D stem cell niche imaging technique (electron and confocal microscopy methods) which allowed the identification of a novel human stem cell niche in the cornea. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact This work has already informed clinical practice by identifying the optimum location from which to harvest an autologous biopsy for patients suitable for cultured limbal epithelial stem cell therapy which has the potential to restore vision. 
 
Description Rapid automation of fabrication of tissues - cornea 
Organisation University College London
Department Institute of Orthopaedics and Musculoskeletal Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Characterisation of the human limbal stem cell niche has led to the award of further funding to my team and our collaborators (Robert Brown, UCL and The Automation Partnership) via the TSB and ESPRC.
Collaborator Contribution Use of technology - rapid automation of fabricated tissues
Impact International conference abstracts submitted. Publication in the journal Biomaterials (Levis et al 2010)
Start Year 2008
 
Description Aniridia Europe meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Patient groups met with the Aniridia Europe Scientific Committee, of which I am a member, to discuss with us plans for future research programmes.

The patient groups are included and play an integral role in a formal strategy that will determine the direction of a pan-European scientific collaboration to understand aniridia and develop new therapies.
Year(s) Of Engagement Activity 2012
 
Description Exhibition and discussion at the Dana Centre, Science Museum, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 'Bonsai Cells' workshop at the BioPlay exhibition at the Dana Centre, The Science Museum, London. Collaboration with Susana Soares, Material Beliefs, Goldsmiths, University of London. 'Bonsai Cells' explored how the shape, network and colour of stem cells contribute to their efficiency in regeneration and distinguish them from diseased cells. Also how stem cell characteristics may be utilised in the production of future renewable plastics. Visitors to the workshop were challenged to consider the benefits and dangers of designers engaging with medical science and were asked to explore their own reaction to a future of control and manipulation of cell patterns and shapes. 28th October 2008

Positive feedback from the venue after the event. Members of the public appreciated the opportunity to question scientists about their research. Our project generated lots of questions and the groups visiting our exhibition had to be encouraged to move on to the next station.
Year(s) Of Engagement Activity 2008
 
Description Public exhibition at the Royal Society, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Collaboration with Susana Soares, Material Beliefs, Goldsmiths University, resulted in a public exhibition of the work of Cells for Sight (including this project) in 'Crossing Over - Exchanges in Art and Biotechnologies' at the Royal Institution of Great Britain, October - November 2008. Funded by the EPSRC

Positive feedback fro the venue. Members of the public were pleased to have access to scientific data through a design concept.
Year(s) Of Engagement Activity 2008
 
Description RNIB, Leeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Gave an oral presentation on the potential for limbal stem cell therapy to restore vision. Explained MRC funded research project (and others) in layman's terms in the context of how it may benefit therapy optimisation.

Patients and general public appreciated the opportunity to ask questions of a scientist.
Year(s) Of Engagement Activity 2008
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I was invited to speak at the Science Society of the Haberdasher Aske's School for Girls. 50 students attended. We had a lively discussion after the talk which also included giving career advise about how to embark upon a career in stem cell research (in response to student questions).

This activity occurred 1 week ago so it remains to be seen what the long-term impact may be. However, a number of the girls who had already decided on a career in science were very interested to know how to get into stem cell research.
Year(s) Of Engagement Activity 2010
 
Description Television interview (BBC Look North 2012) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact News item on BBC Look North covering our research into aniridia.

Raised the profile of our work and public awareness of this rare disease.
Year(s) Of Engagement Activity 2012
 
Description UKNSCN public engagement meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Approximately 200 people attended the event. Four speakers presented their research. This was followed by a panel discussion in response to questions from the audience.

Positive feedback from the UKNSCN after the event.
Year(s) Of Engagement Activity 2011