Alex J Shortt | University College London (original) (raw)

Papers by Alex J Shortt

Progress in retinal and eye research, Jan 23, 2018

Neurotrophic Keratopathy (NK) refers to a condition where corneal epitheliopathy leading to frank... more Neurotrophic Keratopathy (NK) refers to a condition where corneal epitheliopathy leading to frank epithelial defect with or without stromal ulceration (melting) is associated with reduced or absent corneal sensations. Sensory nerves serve nociceptor and trophic functions, which can be affected independently or simultaneously. Loss of trophic function and consequent epithelial breakdown exposes the stroma making it susceptible to enzymatic degradation. Nerve pathology can range from attrition to aberrant re-generation with corresponding symptoms from anaesthesia to hyperaesthesia/allodynia. Many systemic and ocular conditions, including surgery and preserved medications can lead to NK. NK can be mild (epithelium and tear film changes), moderate (non-healing epithelial defect) or severe (stromal melting and perforation). Moderate and severe NK can profoundly affect vision and adversely impact on the quality of life. Medical management with lubricating agents from artificial tears to s...

Doctoral Thesis Ucl, Nov 1, 2009

Abstract Transplantation of ex-vivo cultured limbal epithelial cells (LEC) is an established trea... more Abstract Transplantation of ex-vivo cultured limbal epithelial cells (LEC) is an established treatment for total limbal stem cell deficiency. However, this therapy has preceded the scientific understanding of limbal epithelial stem cells (LESC), the LESC niche and the ...

Investigative Opthalmology & Visual Science, 2014

Investigative Opthalmology & Visual Science, 2015

Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem... more Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behavior, and long-term fate after transplantation. The aim of this research was to evaluate, in vitro, quantum dot (Qdot) technology as a tool for tracking transplanted HLECs. In vitro cultured HLECs were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony-forming efficiency assays and expression of CK3, P63alpha, and ABCG2. Sheets of cultured HLECs labeled with Qdot nanocrystals were transplanted onto decellularized human corneoscleral rims in an organ culture model and observed to investigate the behavior of transplanted cells. Quantum dot labeling had no detrimental effect on HLEC viability or function in vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks after transplantation in our organ culture model. In addition, it was possible to label and observe epithelial cells in intact human corneas by using the Rostock corneal module adapted for use with the Heidelberg HRA. This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks after transplantation; however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterization in in vivo models are required.

Acta Biomaterialia, 2015

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured hu... more Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The 'optimal' RAFT TE was thin, transparent but still handleable and was produced using 0.6 ml of 3 mg/ml collagen. Degradation rates of the 'optimal' RAFT TE and HAM were similar. hLE achieved confluency on 'optimal' RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63a. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency.

Current Eye Research, 2014

Aim of the study: To develop a clinical grade fibrin gel for the culture of oral mucosal epitheli... more Aim of the study: To develop a clinical grade fibrin gel for the culture of oral mucosal epithelial cells (OMEC) intended for ocular surface reconstruction in the treatment of limbal stem cell deficiency (LSCD). Materials and methods: Transparent fibrin gels composed of fibrinogen and thrombin were developed for the culture of epithelial cells. Oral mucosa was harvested from the buccal region of healthy volunteers and cultured as explants on fibrin gels. Tranexamic acid (TA), a clinically approved anti-fibrinolytic agent was added to prevent the fibrin gel from digesting due to cellular activity. The gels were stained for p63a (as a marker of poorly differentiated epithelial cells), CK19, CK13 and CK3 (expressed by OMEC). Epithelial cell stratification was observed using hematoxylin-eosin staining. Results: Addition of TA prevented gels from dissolving during the culture period. OMEC proliferated on the fibrin gel and attained confluence over a 2-week period (±2 d) and exhibited a typical epithelial, cobblestone morphology. Basal OMEC exhibited positive staining for p63a while the superficial cells exhibited positive staining for CK3. The cells expressed a strong immunoreactivity for CK19 and CK13 suggesting that they retained a normal oral epithelial phenotype. Conclusion: Fibrin gels, maintained in the presence of TA, to control the rate of substrate degradation, provide a more robust yet transparent substrate for the culture and transplantation of cultured OMEC. The fibrin gels are easily standardized, the components commercially available, and produced from clinically approved materials. The resulting stratified OMEC-derived epithelium displays characteristics similar to that of a human cornea, e.g. CK3 expression. The conventional dependence on a murine feeder layer for support of epithelial cells is unnecessary with this technique and hence, provides for an attractive alternative for treatment of LSCD.

Secker, G and Watson, SL and Shortt, AJ and Grotendorst, GR and Schultz, GS and Khaw, PT and Dani... more Secker, G and Watson, SL and Shortt, AJ and Grotendorst, GR and Schultz, GS and Khaw, PT and Daniels, JT (2005) Effects of CTGF on HCE re-epithelialisation and migration. In:(Proceedings) Investigative Ophthalmology and Visual Science.(pp. 2139-?).

Acta Ophthalmologica, 2013

ABSTRACT Stem cells contain intrinsic mechanisms that control their behaviour but the environment... more ABSTRACT Stem cells contain intrinsic mechanisms that control their behaviour but the environment immediately surrounding them, known as the niche, also plays a fundamental role. In a host of tissues and organisms the stem cell niche has been shown to maintain stem cells in an undifferentiated state and to regulate their behaviour. This stem cell – niche principle is typified by the corneal limbus. When limbal epithelial stem cells (LESC) are dissociated from their niche in the limbal palisades of Vogt they rapidly differentiate and cease to function as stem cells. It might be imagined that an understanding of the LESC niche would be of the highest priority for researchers in this field but the existence of a surrogate niche, in the form of growth arrested 3T3 fibroblast feeder layers, has allowed research on LESC biology to progress in the absence of a thorough understanding of their native niche. Thankfully LESC niche research has been gathering momentum and several exciting developments hold promise for limbal stem cell deficient patients. Amongst these are the characterisation of the physical structure of the niche and the identification of mesenchymal niche cells, which themselves displaying stem cell properties.

Stem cells translational medicine, 2014

Limbal stem cell deficiency (LSCD) is an eye disorder in which the stem cells responsible for for... more Limbal stem cell deficiency (LSCD) is an eye disorder in which the stem cells responsible for forming the surface skin of the cornea are destroyed by disease. This results in pain, loss of vision, and a cosmetically unpleasant appearance. Many new treatments, including stem cell therapies, are emerging for the treatment of this condition, but assessment of these new technologies is severely hampered by the lack of biomarkers for this disease or validated tools for assessing its severity. The aims of this study were to design and test the reliability of a tool for grading LSCD, to define a set of core outcome measures for use in evaluating treatments for this condition, and to demonstrate their utility. This was achieved by using our defined outcome set (which included the Clinical Outcome Assessment in Surgical Trials of Limbal stem cell deficiency [COASTL] tool) to evaluate the 3-year outcomes for allogeneic ex vivo cultivated limbal epithelial transplantation (allo-CLET) in patients who had bilateral total LSCD secondary to aniridia or Stevens-Johnson syndrome. The results demonstrate that our new grading tool for LSCD, the COASTL tool, is reliable and repeatable, and that improvements in the biomarkers used in this tool correlate positively with improvements in visual acuity. The COASTL tool showed that following allo-CLET there was a decrease in LSCD severity and an increase in visual acuity up to 12 months post-treatment, but thereafter LSCD severity and visual acuity progressively deteriorated.