Cells for Sight Transplantation
& Research Programme

Divisions of Pathology and Cell Biology

Mission
‘To understand the biology and therapeutic potential of stem cells (and the cells with which they interact) in order to develop and deliver novel cell-based therapies to patients suffering from blinding ocular surface disorders’.

Background
Stem Cells
Perhaps the best example of tissue regeneration in the animal kingdom is that of the newt which has the remarkable ability to grow a whole new limb if required. As humans we do not have the capacity for this level of tissue regeneration; however we can replace tissue lost during normal wear and tear such as our hair and epidermis. We can also initiate a tissue repair response following trauma. Provided that the initial injury is not too severe we can even replace parts of our liver, small intestine and blood cells. Our ability to replace these tissues largely rests with a relatively small population of stem cells which have the capacity to self-renew and differentiate along specified molecular pathways throughout life.

Adult stem cells have now been identified throughout the human body. What these amazing, and often controversial, cell populations can teach us will significantly enhance our understanding of cellular and tissue processes in health and disease.

Stem Cells in the Cornea
In order to study adult stem cells we have selected the cornea as a model system. Due to the unique location of a readily accessible source of adult stem cells in a transparent tissue, the cornea provides the opportunity to manipulate stem cells in vitro and to observe them in their niche in vivo more easily than any other body site.

Location of Limbal Epithelial Stem Cells
The corneal limbus is the transition zone between the corneal and neighbouring conjunctival epithelium. The palisades of Vogt in the limbus are thought to provide limbal epithelial stem cells, which maintain the corneal epithelium, with a niche or home. This is based on evidence demonstrating the existence of slow-cycling cells in the basal layer of limbal epithelium which can retain tritiated thymidine label for extended periods of time. In addition, cultured limbal basal cells have the highest proliferative capacity in vitro, surgical removal of the limbus results in delayed healing with non-corneal epithelium and limbal transplants in patients regenerate corneal-like epithelium. The unique location of limbal epithelial stem cells at the limbus conveniently allows them to be studied millimetres away from their differentiated progeny.

The area indicated by the arrow on the left is the limbus. On the right, the palisades of Vogt can be seen in the magnified image. Limbal epithelial stem cells in this region give rise to daughter transient amplifying which migrate and proliferate to form the multilayered epithelium of the cornea. Ultimately these cells terminally differentiate and are sloughed from the surface during normal blinking.

Limbal Epithelial Stem Cell Failure in Patients
Limbal epithelial stem cell failure in patients can be caused by a variety of injuries and diseases including alkali burn, aniridia and Stevens Johnson Syndrome. Owing to a partial or total depletion of limbal epithelial stem cells, the neighbouring conjunctival epithelium migrates over the surface of the cornea causing vascularisation, recurrent epithelial erosion, pain and ultimately blindness.

On the left, a clear, healthy cornea provides a window to the world. On the right, limbal stem cell deficiency in the cornea, causing pain and loss of vision.

Current Research

‘Characterisation of the corneal limbal epithelial stem cell niche’

Collaboration with Mr Stephen Tuft, Prof Fred Fitzke, Dr Peter Munro and Prof Fiona Watt.

We are investigating the properties of the limbal epithelial stem cell niche using a variety of techniques including confocal microscopy and scanning electron microscopy. We are comparing our ex-vivo data with confocal images gathered from human volunteers using the newly developed Rostock attachment for the Heidelberg Retinal Tomograph II (HRT-II) confocal ophthalmoscope.

The left photograph shows a confocal image of the limbus in a whole mounted ex vivo human cornea. The equipment in the centre picture is the Rostock attachment for the Heidelberg Retinal Tomograph II confocal ophthalmoscope which can deliverdetailed images of the limbus in vivo in humans as illustrated in the photograph on the right.

We have optimised the culture and serial propagation of clonal populations of human limbal epithelial stem cells which will allow us to study the effects of specific stimuli on properties and function of these cells.

The large colony in the centre of this photograph has been derived from a single limbal epithelial stem cell. The cells surrounding it are growth arrested 3T3 fibroblasts feeder cells.Over time the stem cell progeny differentiate (arrowed in centre photograph) and eventually form multilayered epithelium (right hand photograph).

‘Characterisation of limbal epithelial stem cells and gene therapy optimisation in a model of congenital aniridia’

Collaboration with Prof Robin Ali.

Aniridia is a congenital disease characterised by mutations in the developmental gene pax-6. Amongst other ocular problems, aniridia causes limbal epithelial stem cell failure in children and is often the cause of premature blindness in these patients. We are using a model of pax-6 haploinsufficiency, which closely resembles the human disease, to characterise limbal epithelial stem cell function and ultimate failure in this disease. Our long-term goal is to combine gene and stem cell therapy to correct genetic defects in children and hence prolong their vision.

‘An ocular surface nutrient medium for dry eye and persistent epithelial defect; in vitro and in vivo studies’

Collaboration with Mr John Dart

We have assisted Mr John Dart in the development of a therapeutic ocular surface medium (TOSM) for the treatment of dry eye and persistent epithelial defect. This therapy has been evaluated both in the laboratory and via clinical trials at Moorfields Eye Hospital.

‘Pre-clinical development of a novel engineered surface for the culture and transplantation of limbal epithelial stem cells’

Collaboration with Prof Sheila MacNeil.

We are working to develop an alternative to human amniotic membrane for the culture and transplantation of limbal epithelial stem cells in patients. The popular use of amniotic membrane is not suitable for all patients as it is an opaque substrate.

‘Characterisation and optimisation of the culture of limbal epithelial stem cells’

Collaboration with Mr Chris Mason.

This project aims to optimise the culture of limbal epithelial stem cells both for therapeutic application and for in vitro research. Currently the optimal methods for culturing these cells involve the use of animal derived-products including bovine serum and often murine 3T3 feeder cells. By defining the culture process we aim to reduce current culture to culture variability and then use the system to better characterise and manipulate limbal epithelial stem cells in vitro.

‘Novel strategies in ocular mucous membrane pemphigoid (MMP): relative contributions of inflammation, T cells and fibroblasts in conjunctival fibrosis’

Collaboration with Mr John Dart and Dr Virginia Calder.

This project is investigating the mechanisms of interaction between inflammatory cells and fibroblasts which lead to fibrosis in mucous membrane pemphigoid in the cornea. In addition, the efficacy of a novel treatment strategy is under evaluation in patients at Moorfields Eye Hospital.

‘The role of CTGF in corneal wound healing’

Collaboration with Prof Greg Schultz.

We are investigating the role of connective tissue growth factor (CTGF) in corneal wound healing. We are particularly interested in the way in which CTGF mediates the effects of the highly pro-scarring protein transforming growth factor beta (TGFbeta). We have recently found that CTGF is required for TGF?-stimulated collagen matrix contraction by fibroblasts and also the conversion of fibroblasts into myofibroblasts.

Using fibroblast populated collagen lattices we discovered that CTGF is required for the conversion of TGF?-stimulated fibroblast to myofibroblast differentiation. This is demonstrated here by the presence of alpha smooth muscle actin fibres in the myofibroblasts in ‘A’. In the presence of antisense oligodeoxynucleotides to CTGF, TGF? was unable to induce myofibroblast differentiation in ‘D’.

Stem Cell Therapy – Cells for Sight Tissue Bank

In order to rapidly translate our research work into sight saving therapies, we have worked closely with our colleagues at Moorfields Eye Hospital, in particular Mr Stephen J. Tuft, to establish ‘The Cells for Sight Tissue Bank’ in the Division of Pathology, Institute of Ophthalmology. The Cells for Sight Tissue Bank was the first if it’s kind to be awarded ‘Tissue Bank Accreditation’ by the Medicines and Healthcare products Regulatory Agency (MHRA) in April 2005.

Cells for Sight Tissue Bank MHRA Accreditation Certificate

This facility, which includes a state-of-the-art class 100 cleanroom, is operated in accordance with Good Manufacturing Practice. This involves working in compliance with a strict quality management system designed to produce cellular therapies to the highest possible standards of quality.

The Cells for Sight Tissue Bank Class 100 Cleanroom

The first cellular therapies we are delivering to patients include both autologous and allogeneic limbal epithelial stem cells cultured on cryopreserved human amniotic membrane. The aim of this therapy is to restore vision and ocular surface comfort in patients suffering from limbal epithelial stem cell deficiency.

The clinical limbal stem cell transplantation research, which began in August 2005, should provide the highest quality clinical data as a direct result of producing the stem cell cultures under GMP, which significantly reduces variation in production and delivery of the therapy, and the large numbers of patients available for study at Moorfields Eye Hospital. We will now be able to produce more accurate data regarding limbal epithelial stem cell therapy efficacy in different patient groups than has previously been possible.

The Future

There remain large obstacles to overcome before many stem cell therapies will enter routine clinical practice. Our future challenges for the understanding the biology and therapeutic potential of stem cells in the eye include; positive identification of stem cells which currently have no specific marker, understanding better the required niche environment for transplanted stem cell survival, identifying alternative sources of stem cells in the body (preferably autologous) for human transplantation in the eye, developing safe animal product free culture systems suitable for therapeutic use, evaluating the potential of embryonic stem cells, combining gene therapy with stem cell and other cellular therapies for the treatment of corneal dystrophies, overcoming the problems associated with allogeneic stem cell transplantation in the cornea, developing methods to label and visually track stem cells in humans using the cornea as a model, resolving ethical issues surrounding the use of embryonic stem cell lines and finally compliance of any potential stem cell therapy to a rapidly changing regulatory environment. Resolution of the argument that adult stem cells can transdifferentiate to produce cells of different lineages and understanding why if pluripotent stem cells do circulate throughout the adult human body why they do not reliably target sites of injury and initiate regeneration of damaged tissue are also interesting questions to be addressed. Successfully addressing these issues in the cornea will not only help patients suffering from blinding ocular surface diseases, but will also further our understanding of stem cells in general. Owing to its unique optical properties and readily accessible location, we anticipate that the cornea will unlock many of the secrets of stem cell function, regulation and survival in the body. This in turn will contribute to the goal of regenerative medicine which is to replace abnormal tissue repair following from injury and disease with normal tissue regeneration and function.

Cells for Sight Team

Head of Group
Dr Julie T. Daniels, PhD
Lecturer, Epithelial Stem Cell Biology, Institute of Ophthalmology
Director, Cells for Sight Tissue Bank, Moorfields Eye Hospital
j.daniels@ucl.ac.uk

Team
Mrs Joanne Daniels, BSc (Hons)
MEH Special Trustees Cells for Sight Quality Assurance Officer

Mr Laurence Dufaur, BSc (Hons)
MEH Special Trustees Stem Cell Transplantation and Research Technician
l.dufaur@ucl.ac.uk

Ms Anna Harris, MSc
EPSRC EngD Student
‘Characterisation and optimisation of the culture of limbal epithelial stem cells’
anna.harris@ucl.ac.uk

Dr Maria Notara, PhD
BBSRC Postdoctoral Research Fellow
‘Pre-clinical development of a novel engineered surface for the culture and transplantation of limbal epithelial stem cells’
m.notara@ucl.ac.uk

Ms Valerie Saw, FRANZCO
MEH Special Trustees Clinical Research Fellow
‘Novel strategies in ocular mucous membrane pemphigoid (MMP): relative contributions of inflammation, T cells and fibroblasts in conjunctival fibrosis’
valerie.saw@moorfields.nhs.uk

Ms Genevieve Secker, BSc (Hons)
Eranda Foundation PhD Student
‘Characterisation of limbal epithelial stem cells and gene therapy optimisation in a model of congenital aniridia’
g.secker@ucl.ac.uk

Mr Alex Shortt,, MRCOphth
MRC Clinical Research Training Fellow
‘Characterisation of the corneal limbal epithelial stem cell niche’
a.shortt@ucl.ac.uk

Alumni
Ms Stephanie Watson, FRANZCO
‘An ocular surface nutrient medium for dry eye and persistent epithelial defect; in vitro and in vivo studies’.

Collaborators

Prof Robin Ali, Institute of Ophthalmology
Prof Oya Alpar, School of Pharmacy, University of London
Prof Robert Brown, Institute of Orthopaedics
Dr Virginia Calder, Institute of Ophthalmology
Mr John Dart, Moorfields Eye Hospital
Prof Fred Fitzke, Institute of Ophthalmology
Prof Peng T. Khaw, Institute of Ophthalmology
Prof Sheila MacNeil, University of Sheffield
Mr Chris Mason, Dept of Biochemical Engineering, UCL
Dr Peter Munro, Institute of Ophthalmology
Prof Gill Murphy, University of Cambridge
Prof Santa Ono, Institute of Ophthalmology
Prof Gregory S. Schultz, University of Florida
Mr Stephen J. Tuft, Moorfields Eye Hospital
Prof Fiona Watt, Cancer Research UK

Funding
The group has received funding from:

BBSRC
Department of Health
ERANDA Foundation
Fight for Sight
Hayman Trust
Helen Hamlyn Trust
MRC
RNIB
Special Trustees of Moorfields Eye Hospital

Publications

1. Limb, GA, Daniels, JT, Cambrey, A, Secker, GA, Shortt, AJ, Lawrence, JM and Khaw. 2006 Current prospects for adult stem cell-based therapies in ocular repair and regeneration. Curr. Eye Res. In Press

2. Garrett, G, Khaw, PT, Blalock, TD, Grotendorst, GR, Schultz, GS and Daniels, JT. 2004 Connective tissue growth factor dependency of TGF-1-stimulation of myofibroblast differentiation and collagen matrix contraction in the presence of mechanical stress. Invest. Ophthalmol. Vis. Sci. 45 (4): 1109-1116.

3. Wong, TT, Daniels, JT, Crowston, JG and Khaw, PT. 2004 MMP inhibition prevents human lens epithelial cell migration and contraction of lens capsule. Br. J. Ophthalmol. 88(7): 868-872.

4. Crowston, JG, Chang, LH, Daniels, JT, Khaw, PT and Akbar, AN. 2004 T lymphocyte-mediated lysis of mitomycin-C treated Tenon’s capsule fibroblasts. Br. J. Ophthalmol. 88 (3): 399-405.

5. Khaw, PT, Clarke, JCK, Mead, AL, Wong, TTL, Cambrey, A and Daniels, JT. 2004 Controlling tissue repair and regeneration after surgery: new treatments and techniques. In: Glaucoma therapy current issues and controversies. Eds. Shaarawy, T and Flammer, J. Chapter 22: 291-309.

6. Daniels, JT, Geerling, G, Alexander, RA, Murphy, G, Khaw, PT and Saarialho-Kere, U. 2003 Temporal and spatial expression of matrix metalloproteinases during wound healing of human cornea. Exp. Eye Res. 77(6): 653-664.

7. Daniels, JT, Schultz, GS, Blalock, Grotendorst, GR, Dean, NM and Khaw, PT. 2003 Mediation of TGF1-stimulated matrix contraction by fibroblasts: a role for CTGF in contractile scarring. Am. J. Path. 163 (5): 2043-2052.

8. Daniels, JT, Limb, GA, Saarialho-Kere, U, Murphy, G. and Khaw, PT. 2003 Human corneal epithelial cells require MMP-1 for HGF-mediated migration on collagen I. Invest. Ophthalmol. Vis. Sci. 44: 1048-1055.

9. Daniels, JT, Cambrey, AD, Occleston, NL, Garrett, Q, Tarnuzzer, RW, Schultz, GS and Khaw, PT. 2003 Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and production in vitro. Invest. Ophthalmol. Vis. Sci. 44: 1104-1110.

10. Wong, TTL, Sethi, C, Daniels, JT, Limb, GA, Murphy, G and Khaw, PT. 2002 Matrix metalloproteinases in disease and repair processes in the anterior segment. Survey Ophthalmol. 47 (3): 239-256.

11. Occleston, NL, Daniels, JT and Khaw, PT. 2002 Wound Healing: Laboratory Investigation and modulating agents. In: An Introduction to Vascular Biology: from Basic Science to Clinical Practice. Eds. Hunt, B.J., Poston, L., Schachter, M. and Halliday, A. Cambridge University Press. Chapter 7: 129-165.

12. Limb GA, Daniels JT, Pleass RD, Blood I, Murphy G, Charteris DG, Luthert PJ and Khaw PT. 2002 Expression of MMP-2 and MMP-9 by retinal Muller Cells: Modulation by extracellular matrix-bound TNF. Am. J. Path. 160 (5): 1847-1856.

13. Khaw, PT, Cambrey, AD, Limb, GA and Daniels, JT. 2002 Gene therapy: new ‘magic bullets’ to prevent ocular scarring. Br. J. Ophthalmol. 86 (5): 490-492.

14. Crowston, JG, Chang, LH, Constable, PH, Daniels, JT, Akbar, AN and Khaw, PT. 2002 Apoptosis gene expression and death receptor signalling in mitomycin-C-treated human Tenon’s capsule fibroblasts. Invest. Ophthalmol. Vis. Sci. 43 (3): 692-699.

15. Daniels, JT, Dart, JKG, Tuft, SJ and Khaw, PT. 2001 Corneal stem cells in review. Wound Repair and Regeneration. 9 (6): 483-494.

16. Khaw, PT, Chang, L, Wong, TT, Mead, A, Daniels, JT, and Cordeiro, MF. 2001 Modulation of wound healing after glaucoma filtration surgery. Curr. Opin. Ophthalmol. 12(2): 143-148.

17. Geerling G, Daniels JT, Dart JKG, Cree IA, and Khaw PT. 2001 Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 42 (4): 948-956.

18. Poon, AC, Geerling G, Dart, JKG, Fraenkel, GE and Daniels, JT. 2001 Autologous serum eye drops for dry eyes and epithelial defects: clinical and in-vitro toxicity studies. Br. J. Ophthalmol. 85 (10): 1188-1197.

19. Stephens, P, Davies, KJ, Occleston, NL, Pleass, RD, Kon, CH, Daniels, JT, Khaw, PT and Thomas, DW 2001 Phenotypic differences between oral mucosal and skin fibroblasts in the expression, production and activity of matrix metalloproteinases and their inhibitors in an in vitro model of extracellular matrix reorganisation. Br. J. Dermatol 144 (2): 229-237.

20. Daniels, JT and Khaw, PT. 2000 Temporal stimulation of corneal fibroblast wound healing activity by differentiating epithelium in vitro. Invest. Ophthalmol. Vis. Sci. 41 (12): 3754-3762.

21. Cordeiro, MF, Chang, L, Lim, KS, Daniels, JT, Pleass, RD, Sirawardena, D and Khaw, PT. 2000 Modulating conjunctival wound healing. Eye 14 (3): 536-547.

22. Geerling, G, Joussen, AM, Daniels, JT, Mulholland, B, Khaw, PT and Dart, JK. 1999 Matrix metalloproteinases in sterile corneal melts. Ann. NY Acad. Sci. 878: 571-574.

23. Daniels, JT, Occleston, NL, Crowston, JG and Khaw, PT. 1999 Effects of antimetabolite induced cellular growth arrest on fibroblast-fibroblast interactions. Exp. Eye Res. 69: 117-127.

24. Daniels, JT, Occleston, NL, Crowston, JG, Cordeiro, MF, Alexander, RA, Wilkins, M., Porter, R., Brown, R.A. and Khaw, P.T. 1998 Understanding and controlling the scarring response: the contribution of histology and microscopy. Microscopy Research and Technique 42 (5): 317-333.

25. Crowston, JG, Akbar, AN, Constable, PH, Occleston, NL, Daniels, JT and Khaw, PT. 1998 Antimetabolite induced apoptosis in Tenon’s capsule fibroblasts. Invest. Ophthalmol. Vis. Sci. 39 (2): 449-454.

26. Occleston, NL, Daniels, JT, Tarnuzzer, RW, Sethi, KK, Alexander, RA, Bhattacharya, SS and Khaw, PT. 1997 Single exposures to antiproliferatives: long term effects on ocular fibroblast fibroblast wound healing behaviour. Invest. Ophthalmol. Vis. Sci. 38 (10): 1998-2007.

27. Kon, CH, Occleston, NL, Charteris, D, Daniels, J, Aylward, GW and Khaw, PT. 1998 A prospective study of matrix metalloproteinases in proliferative vitroretinopathy. Invest. Ophthalmol. Vis. Sci. 39 (8): 1524-1529.

28. Daniels, JT, Kearney, JN and Ingham, E. 1997 An investigation into the potential of extracellular matrix factors for attachment and proliferation of human keratinocytes in skin substitutes. Burns 23 (1): 26-31.

29. Daniels, JT, Kearney, JN and Ingham, E. 1996 Human keratinocyte isolation and cell culture: a survey of current practices in the UK. Burns 22 (1): 35-39.

30. Daniels, JT, Harris, IR, Kearney, JN and Ingham, E. 1995 Calcium: a crucial consideration in serum-free keratinocyte culture. Exp. Dermatol. 4 (4): 183-191.

This page last modified 27 October, 2006 by David Daniel