Modulation of limbal niche stiffness to regulate stem cell differentiation

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.

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).

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).