Therapeutic corneal stem cell delivery using hydrogels without the need for ex vivo expansion

The cornea is our window to the world, once compromised by wounding, disease or age, a loss of vision results. By providing new methods of corneal stem cell transplantation the sight of many more patients can be restored. Currently, corneal stem cell transplantation involves a lengthy pre-surgical step during which time the corneal stem cells are harvested from donor eyes then grown in a flask to increase their number prior to transplantation. This step is time consuming, technically demanding and hugely expensive as it requires highly trained surgeons/scientists and specialized laboratories. If the potential for corneal stem cell therapy is to be fully realised cost effective surgical procedures need to be developed urgently. This investigation stems from our previous work in understanding the limitations of the current corneal stem cell transplantation techniques, specifically the materials used to grow and convey the stem cells to the patient. Previously, we have quantified the nanostructure of corneal stem cell substrates and applied these measurements to the design of new biomaterials. We have since discovered a gel-based substrate that encapsulates corneal stem cells has significant advantages over current biomaterials. Therefore, to simplify corneal stem cell transplantation we will develop a gel-based stem cell delivery system that can transport and retain a relatively small number of freshly isolated corneal stem cells to the surface of the eye using a range of experimental models that are close to humans. The gel will retain the transplanted stem cells long enough for them to attach, grow and repair the damaged cornea. The gel will be over 90% water (a hydrogel) and possess a degree of flexibility very similar to the host tissue. Unlike other forms of corneal stem cell transplant the donor stem cells will not require extended periods in culture. Therefore, preparation of the transplant material will take minutes not weeks to complete and the transplanted cells will not contain animal derived growth factors or potentially dangerous unwanted cell types (both unavoidable consequences of growing stem cells outside the body). Specific small molecules that can stimulate the cells to grow may also be added to the gel aiding the replacement of xenobiotic materials.
We believe the application of hydrogels to the delivery of therapeutic stem cells will also be applicable to other diseased tissues (skin, bone, brain, heart) where there is a need to localise a small number of transplanted stem cells in a very precise manner.

The research is aimed at developing preclinical studies for the design of cell based therapies to treat total limbal stem cell deficiency, a condition characterized by damage to the corneal epithelial stem cells and which constitutes a common cause of severe vision impairment and blindness. The proposed investigation stems from our previous work in understanding the limitations of the current vehicles used in corneal stem cell transplantation and our recent work in developing alternatives to these vehicles. Previously, we have quantified the nanostructure of amniotic membrane (current vehicle of choice for corneal stem cell transplantation) and are applying these observations to the design of a new biomaterial to replace amniotic membrane. During these studies we have discovered that a gel based vehicle that encapsulates the donor corneal stem cells prior to therapeutic delivery has many advantages over the current solid-support type vehicles typified by amniotic membrane and its mimics. To progress our work towards the translational stage, we aim to validate these findings using a suite of experimental models that are close to humans. We will therefore investigate whether transplantation of corneal stem cells within a gel can remain viable, have a good bio-adhesion and restore corneal clarity in the rabbit eye following wounding. We have isolated corneal stem cells from human, bovine and rabbit and are in the position to propagate these cells for autologous and/or allogeneic transplantation by using a model of the human ocular surface, a bovine organ culture and a rabbit model respectively. The main objectives of our proposed research i) to isolate and propagate limbal stem cells and to differentiate these cells to form a functional ocular surface within a natural or synthetic hydrogel; ii) to develop hydrogels with improved bio-adhesive properties and the ability to gel in situ; iii) to assess the role of PI3Ks in corneal epithelial cell differentiation and proliferation by specific inhibition/activation and to incorporate small molecule inhibitors of PI3K regulatory molecules into the gel with a view to improving transplanted cell growth; iv) to generate a functional corneal epithelium from transplanted stem cells in a rabbit without the need for ex vivo expansion or supplemental growth factors. We expect that the results obtained from this study would advance this research into an immediate translational stage towards the development of a safe and simple one step therapy to treat total limbal stem cell deficiency.