Modulation of limbal niche stiffness to regulate stem cell differentiation

Lead Research Organisation: Newcastle University
Department Name: Institute of Human Genetics

Abstract

Tissue engineering is a newly emerging biomedical methodology to assist and accelerate the regeneration of defective and damaged tissues based largely on the natural healing potentials of stem cells. For this new therapeutic strategy, it is indispensable to provide cells with a local environment (niche) that enhances and regulates their proliferation and differentiation. In this regard, biomaterial technology currently leads the way in trying to fulfil these requirements by recreating artificial three-dimensional environments. However, understanding how, why and in what environment these cells differentiate in a lineage-specific manner are essential for tissue engineering and stem cell therapy. We believe that this underlying biology (i.e. specific signals or stimuli which make the cells respond) has not yet been properly investigated and that this is likely to undermine current attempts to recreate the stem cell niche using biomaterials. Therefore, we plan to understand the role substrate stiffness plays in maintaining the corneal stem cell niche (the home for cells required to cover the surface of the cornea). Our expertise in ocular surface models means we are well placed to investigate the changes in gene and protein expression associated with substrate stiffness dependent (mechanosensitive) differentiation. Together these investigations will feed directly into the field of tissue engineering and our own on-going MRC, BBSRC and EPSRC research into ocular surface biomaterials and corneal stem cell transplantation.
Acoustically induced inelastic light scattering (Brillouin microscopy) allows non-contact, direct readout of the mechanical properties of a material and has been used successfully to measure, at microscopic resolution, the biomechanical properties of the cornea and lens of the human eye. We plan to employ this sophisticated method to measure stiffness across the surface of normal and disease/wounded corneas producing high resolution maps of the tissues' mechanical properties. We will then seek to affect localised stiffness using an enzyme (collagenase) to break up the corneas main structural protein (collagen) in a highly controlled manner. The enzyme will be delivered by a gel that sticks to the cornea to allow precise positioning of the enzyme and a corresponding localised effect on stiffness. We have shown previously that corneal stem cells differentiate when grown on stiff collagen gels and that collagenase can reduce this stiffness with a proportional reduction in differentiated cells. Therefore we plan to investigate a new pre-corneal stem cell transplantation technique. In short, having validated that the corneal stem cell niche is sensitive to changes in its stiffness we will develop a method to control its stiffness prior to corneal stem cell transplantation with the belief that this will result in an improved residency time for the transplanted cells in an undifferentiated form. The predicted outcome being a significantly improved success rate for corneal stem cell transplantation. Recently the overall clinical success rate of corneal stem cell transplantation has remained static at approx. 75% with half of those able to discern two lines or more of symbols on an eye chart. Whilst this represents a remarkable surgical achievement there remains considerable room for improvement. We believe restoring the corneal tissues mechanical properties prior to stem cell transplantation represents a significant surgical enhancement.

Technical Summary

We seek to prove that it is largely the mechanical properties (stiffness) of the limbal niche which maintains stem cells in an undifferentiated state and that disease/wounding affects the niche's mechanical properties prohibiting stem cell survival and secondly that we can alter the niche's mechanical properties by precise delivery of collagenase, using a medically approved hydrogel, prior to stem cell therapy. This study is important because a) it is the first to investigate the mechanical properties of a stem cell niche, b) it is the first to consider modification of tissue stiffness as an adjunct to stem cell therapy and c) the anticipated treatment is simple, cheap and applicable to a wide range of stem cell therapies. We have previously shown that the mechanical properties of a collagen substrate can be tuned to enable differentiation of limbal stem cells. Similarly we have found that collagenase can reduce the substrates stiffness in a dose dependent manner producing an improved level of undifferentiated limbal stem cells. We propose to gauge the impact of elastic modulus on limbal stem cell niche function and whether the precise application of collagenase as a surgical pre-treatment can confer a significant improvement to the maintenance of undifferentiated limbal stem cells in culture. The elastic-modulus of the cornea will be measured by the application of acoustically induced inelastic light scattering (Brillouin microscopy) that allows non-contact, direct readout of the corneas mechanical properties. The translational outcome of this proposal would be the attainment of critical safety and efficacy data required for a first-in-man trial of collagenase for the restoration of the limbal stem cell niche (collagenase is already used clinically in skin wound management).

Planned Impact

The proposed programme of work is timely as it fits within a number of strategic objectives recently announced within 'A Strategy for UK Regenerative Medicine'. For example our objectives include improvements to our understanding of cellular differentiation directed by extracellular modification, specifically within the stem cell niche to benefit cell-based therapies via an acellular approach. Furthermore we aim to reach these objectives via use of strong complementary skills, expertise and infrastructure across the disciplines of ophthalmology, cell/matrix biology and photonics.
Non-academic beneficiaries will include the NHS, patients, UK regenerative medicine business and MoD. Ophthalmic surgeons will gain improved predictability of clinical outcome following limbal stem cell therapy and reduced repeat surgeries (lowering cost to NHS). Patients would benefit from improved vision in terms of overall success rates and individual visual acuity (lowering cost of patient support associated with improved vision). This would happen following clinical trials and approximately 5 years from start of grant.
Established healthcare companies can use the technology to move more towards the regenerative medicine space, SME's can use the simplicity (and relative low cost of this acellular approach) to gain traction within the regenerative medicine space. This could be achieved within 3 years of the grant starting. The application of substrate stiffness in bioprocessing would also be evaluated during the 3 year project in partnership with BRIC members. This area of cell based bioprocessing has previously been given little consideration but methods for scaling up production of cells with defined levels of differentiation are urgently required.
The MoD would benefit, specifically in terms of maintaining manpower and reducing morbidity and pain associated with wounded personnel. The use of in situ gelation of a PEG hydrogel as a vehicle or material for improved wound care management could be assessed relatively quickly (within 12months of the project start).

Publications

10 25 50
 
Description We have made the startling discovery that there is a difference in the elastic modulus across the surface of the cornea (sub-epithelial) and that difference is required for epithelial homeostasis. Moreover certain wounds disrupt this difference in elastic modulus and by repairing this wound healing is improved.
Exploitation Route use of proteases to bring about new type of therapy named biomechanical modulation therapy
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Member of MRC Regen Med panel
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Participating in development of new/revised MRC Regen Med grant application. Grant is now shorter and more focussed
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
URL https://www.mrc.ac.uk/funding/browse/biomedical-catalyst-rmrc/biomedical-catalyst-regenerative-medic...
 
Description Improving corneal healing via modulation of tissue biomechanics
Amount £125,000 (GBP)
Organisation Ulverscroft Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 04/2021
 
Description TDRF/Standard
Amount £105,000 (GBP)
Funding ID BB/N021576/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 08/2017
 
Description Understanding the molecular and cellular complexity of human cornea through single cell analyses
Amount £458,770 (GBP)
Funding ID MR/S035826/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2018 
End 12/2020
 
Title High Resolution Brillouin Spectroscopy for imaging tissue biomechanics 
Description Our work so far has focussed on building a High resolution Brillouin microscope that can probe collagen dense tissues or tissue-engineered constructs and measure their stiffness non-destructively (without physical perturbation). To this end, we have successfully built a new instrument and demonstrated its effectiveness in measuring discrete differences in the mechanical properties within biological samples (ranging from 20 µm-thin to 2 mm-thick) at a 1×1×5 µm resolution. The current instrument is capable of quickly generating highly reproducible data, using a novel adaptive optics inferferometric approach to suppress the elastic backscatter to measure Brillouin shifts with 0.01 GHz resolution, and with corresponding mechanical elastic sensitivity results comparable to those obtained in previous studies using different methodologies 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact Our High resolution Brillouin microscope has successfully facilitated a landmark study in which we show that the stem cell niche (within the cornea) is measurably more compliant that tissue outside the niche area and that this change in stiffness is directly associated with the stem cells level of differentiation. Moreover we show that by affecting tissue stiffness in vivo the cells within this area change their phenotype in a predicable manner. This works leads to a new therapeutic avenue. 
 
Title continuous process for production of adherent cells 
Description Adherent cells are normally cultured in batches, all them detaching at once for collection. We have invented a coating which facilitates continuous adhesion then detachment of individual cells (i.e. they do not all detach at once). This now means that the number of adherent cells produced within a tissue culture flask is no longer limited to the area of the flask (to which the cells are adhered) and cell can be cultured within a single flas (or coated area) indefinitely. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact A business plan to commercialise this technology has been written. 
URL https://pubs.acs.org/doi/abs/10.1021/acsami.7b09809
 
Title substrate curvature to direct cell alignment 
Description We have developed a novel approach to create tissue from aligned cells. This was done by making the novel observation that cell align on large macro scaled curved substrates. Using such an approach we have shown that function corneal tissues can be grown from the cells only and that the resulting structure has both shape and function of a normal cornea 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2017 
Provided To Others? Yes  
Impact Led to EU grant application. 
URL http://onlinelibrary.wiley.com/doi/10.1002/adbi.201700135/full
 
Description University of Missouri 
Organisation University of Missouri
Country United States 
Sector Academic/University 
PI Contribution we supplied the experimental design and intellectual prospective
Collaborator Contribution Prof Rajiv Mohan performed a series of animal studies in his lab invistigating the effect of collagenase as a drug for stem cell niche repair
Impact Publication in Nat Comms 2019 reporting on role of substrate stiffness on limbal stem cell niche function
Start Year 2016
 
Title enzymatic sofenting of tissue to affect cell phenotype in vivo 
Description Having shown that the stem cell niche within the cornea is (as hypothesised) is softer than the central cornea. We have performed initial in vivo studies to show that collagenase can soften tissue without causing and adverse effect and moreover it softens the tissues causing the resident cells to become less differentiated or prevents them differentiated. This approach has huge clinical potential for treatment of diseases which the the stem cell niche, as we believe this is partially caused by a stiffening of the tissue within the niche. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Refinement. Clinical
Year Development Stage Completed 2016
Development Status Actively seeking support
Impact currently under 2nd round of review process for leading journal (Nature Materials) 
 
Company Name 3D BIO-TISSUES LIMITED 
Description The Company "3D Bio-Tissues" (3DBT) has identified a significant and unique commercial opportunity in the rapidly developing 3D bio-printing market. We will offer superior, transplantable corneal replicates for the treatment of corneal stromal disorders. These tissue engineered replicates will uniquely conform to the hosts' needs in terms of shape and thickness, providing adequate cells and materials for the restoration of the patients' sight. 
Year Established 2018 
Impact Will employ technology developed by Prof Connon on 3D printing the cornea
 
Description Interviews for national news 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Press release associate with paper describing new way to grow human corneas then led to 2x TV news interviews (BBC and ITV) and BBC Radio NE
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/news/uk-england-tyne-41685982
 
Description Panel member for MRC Regen Med 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Sitting on MRC regend Med panel
Year(s) Of Engagement Activity 2015,2016
 
Description School visit (Tyneside) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talked about being a Professor of Tissue Engineering to class of 8 year olds.
Year(s) Of Engagement Activity 2016