Ex-vivo Cultivated Limbal Stem Cell Transplantation for Treatment of Superficial Corneal Pathologies
Phase: PHASE1
Conditions: Corneal Injuries, Corneal Burns, Corneal Scars and Opacities
Sponsor: L.V. Prasad Eye Institute

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PROFORMA FOR SUBMISSION AND APPROVAL OF RESEARCH PROJECT 
Title of the Project:   
Ex-vivo Cultivated Limbal Stem Cell Transplantation for Treatment of Superficial 
Corneal Pathologies                                               
Objectives of the Project: 
To evaluate the safety of ex-vivo cultivated limbal stromal stem cells for the 
treatment of visually significant superficial corneal stromal scarring and other pathologies. 
Names of the Principal and Co-investigators with designation and qualifications: 
Principal Investigator 
Sayan Basu, MBBS, MS 
Consultant Corneal Surgeon and Scientist, 
L V Prasad Eye Institute,  
Hyderabad, India. 
Co-Investigator 
Virender S Sangwan, MS 
Director, Center for Ocular Regeneration(CORE), 
Director, Srujana-Center for Innovation, 
Dr. Paul Dubord Chair in Cornea, 
L V Prasad Eye Institute, Hyderabad, India. 
Co-Investigator 
Dr. Vivek Singh, Ph.D., (Scientist) 
Prof. Brien Holden Eye Research Center,  
Champalimaud Translational Centre for Eye Research, 
HERF, L V Prasad Eye Institute, 
Hyderabad, India. 
Co-Investigator 
Mukesh Damala, MSc (Biochemistry) 
Prof. Brien Holden Eye Research Center, 
Champalimaud Translational Centre for Eye Research, 
HERF, L V Prasad Eye Institute, 
Hyderabad, India. 
 
Outline of Previous Work in the field with relevant References: 
     Background: 
Keratocytes, normally quiescent cells of the corneal stroma, transform into 
fibroblasts in response to injury or inflammation and repair the damaged cornea by laying 
down scar tissue (1, 2). The presence of altered extracellular matrix components in such 
stromal scars renders the cornea optically opaque, resulting in visual loss (3). Visual 
impairment and blindness due to corneal scarring affects millions worldwide (4, 5) and is the 
commonest indication for corneal transplantation in the developing world (6). Although 
replacing the scarred tissue with a clear corneal allograft is usually effective in improving 
vision (7), the global demand for donor corneal tissue vastly exceeds its availability. 
Moreover, post-operative complications like immune-rejection, astigmatism and infection 
often restrict the functional survival of corneal allografts (8), especially in developing 
countries (9). As such, there is increasing interest in the development of therapeutic 
alternatives to corneal transplant, including stem cell therapy, cell-free collagen scaffolds, 
and bioengineered constructs (10-12).The recent discovery of multi-potent stem cells in the 

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corneal stroma has opened up the possibility of developing a cell-based approach to 
treating corneal scars as an alternative to keratoplasty (13, 14). In a murine model of 
corneal opacity, human stromal stem cells were effective in regenerating normal corneal 
extra-cellular matrix and repairing collagen fibril defects (15). Subsequently other groups 
have independently confirmed the existence of these adult stem cells of mesenchymal 
lineage in the human peripheral corneal and limbal stroma (16-18). These stromal stem cells 
are immune-suppressant and may have a role not only in remodeling but also in preventing 
corneal stromal scars (19).  
As the next step towards translating this research into clinical therapy, we proposed 
to investigate the transplantation of ex-vivo cultivated allogenic limbal stromal cells for the 
treatment of the corneal pathologies. The limbus is an ideal source as the stem cells are 
numerous and located very superficially in the tissue (17). Pre-clinical work suggests human 
corneal stromal stem cells can be isolated from the cadaveric tissues, cultivated in 
conditions suitable for cell based therapy and used to prevent fibrosis in a murine model of 
corneal stromal scarring. Further, these cells are able to successfully engraft, differentiate, 
and mediate wound healing in the corneal stroma such that the tissue remains healthy, free 
of fibrotic tissue, and optically transparent. The clinical implications of these findings are 
substantial in that it represents the potential to lessen the burden on donor tissue necessary 
for corneal allografts by using cultured cells to regenerate tissue. We foresee the ability of a 
clinician to and grow and expand the cells in number and after surgically removing the scar 
tissue from the wounded eye, apply the cultured limbal stem cells to regenerate healthy, 
transparent tissue. 
 
References: 
1. M. L. Funderburgh, M. M. Mann, J. L. Funderburgh, Keratocyte phenotype is enhanced in 
the absence of attachment to the substratum. Molecular vision 14, 308 (2008). 
2. J. V. Jester, A. Budge, S. Fisher, J. Huang, Corneal keratocytes: phenotypic and species 
differences in abundant protein expression and in vitro light-scattering. Investigative 
ophthalmology & visual science 46, 2369 (Jul, 2005). 
3. C. Cintron, J. D. Gregory, S. P. Damle, C. L. Kublin, Biochemical analyses of proteoglycans 
in rabbit corneal scars. Investigative ophthalmology & visual science 31, 1975 (Oct, 1990). 
4. D. Pascolini et al., 2002 global update of available data on visual impairment: a 
compilation of population-based prevalence studies. Ophthalmic epidemiology 11, 67 (Apr, 
2004). 
5. J. P. Whitcher, M. Srinivasan, M. P. Upadhyay, Corneal blindness: a global perspective. 
Bulletin of the World Health Organization 79, 214 (2001). 
6. L. Dandona et al., Indications for penetrating keratoplasty in India. Indian journal of 
ophthalmology 45, 163 (Sep, 1997). 

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7. K. A. Williams et al., How effective is penetrating corneal transplantation? Factors 
influencing long-term outcome in multivariate analysis. Transplantation 81, 896 (Mar 27, 
2006). 
8. D. J. Coster, K. A. Williams, The impact of corneal allograft rejection on the long-term 
outcome of corneal transplantation. American journal of ophthalmology 140, 1112 (Dec, 
2005). 
9. L. Dandona, T. J. Naduvilath, M. Janarthanan, K. Ragu, G. N. Rao, Survival analysis and 
visual outcome in a large series of corneal transplants in India. The British journal of 
ophthalmology 81, 726 (Sep, 1997). 
10. A. A. Chan et al., Differentiation of human embryonic stem cells into cells with corneal 
keratocyte phenotype. Plos one 8, e56831 (2013). 
11. M. Griffith et al., Regenerative approaches as alternatives to donor allografting for 
restoration of corneal function. The ocular surface 10, 170 (Jul, 2012). 
12. J. Wu, Y. Du, S. C. Watkins, J. L. Funderburgh, W. R. Wagner, The engineering of 
organized human corneal tissue through the spatial guidance of corneal stromal stem cells. 
Biomaterials 33, 1343 (Feb, 2012). 
13. Y. Du, M. L. Funderburgh, M. M. Mann, N. SundarRaj, J. L. Funderburgh, Multipotent 
stem cells in human corneal stroma. Stem cells 23, 1266 (Oct, 2005). 
14. Y. Du et al., Secretion and organization of a cornea-like tissue in vitro by stem cells from 
human corneal stroma. Investigative ophthalmology & visual science 48, 5038 (Nov, 2007). 
15. Y. Du et al., Stem cell therapy restores transparency to defective murine corneas. Stem 
cells 27, 1635 (Jul, 2009). 
16. M. J. Branch et al., Mesenchymal stem cells in the human corneal limbal stroma. 
Investigative ophthalmology & visual science 53, 5109 (Aug, 2012). 
17. G. G. Li, Y. T. Zhu, H. T. Xie, S. Y. Chen, S. C. Tseng, Mesenchymal stem cells derived from 
human limbal niche cells. Investigative ophthalmology & visual science 53, 5686 (Aug, 2012). 
18. N. Polisetty, A. Fatima, S. L. Madhira, V. S. Sangwan, G. K. Vemuganti, Mesenchymal cells 
from limbal stroma of human eye. Molecular vision 14, 431 (2008). 
19. L. J. Bray et al., Immunosuppressive properties of mesenchymal stromal cell cultures 
derived from the limbus of human and rabbit corneas. Cytotherapy 16, 64 (Jan, 2014). 
• Brief description of the present work: 
In this prospective interventional study patients with unilateral superficial corneal 
scars will undergo a surgical procedure. Limbal ring from a cadaveric donor tissue, which is 
therapeutically accepted and serologically tested, is collected. This tissue will then be 
cultivated in the stem cell biology laboratory using standardized culture technique. Briefly 
the limbal tissue will be cut up into small pieces and digested overnight using an enzyme 
(Collagenase L). The cells obtained from the digest will be cultured on a petri-dish using 2% 

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serum and growth factors. The cultured cells will be passaged three times to remove all 
epithelial cells from the culture. In the second procedure, the central corneal epithelium will 
be removed using a surgical sponge. 0.1ml of stromal cells in a concentration of 5x103 
cells/uL diluted in the thrombin component of fibrin glue (TISEEL, Baxter) will be applied to 
the debrided corneal stroma. A soft bandage contact lens will be placed over the cornea at 
the end of the procedure. The patient will receive topical antibiotic and steroid eye drops in 
the post-operative period. Periodic comprehensive ophthalmic evaluation along with 
anterior segment optical coherence tomography (ASOCT) scanning and slit-lamp 
photography will be done at day 1, day 7, day 45 and day 90. The primary outcome measure 
of this study is to note any ocular or systemic adverse effects of this intervention at the 
various post-operative time points. The secondary outcome measures are visual 
improvement and change in the density and appearance of the corneal scarring and other 
pathologies after treatment. 
• Justification for use of human volunteers / patients: 
This is a pilot study to ascertain the safety of application of ex-vivo cultivated limbal 
stromal cells in human corneas. Pre-clinical work in murine models have already 
demonstrated efficacy of this technique in curing murine corneal scars.  
 
• Patients - state the type of patients who will be studied and how they will be selected? 
How often and how they will be selected? How often and how many will be studied? 
Which consultant(s) will be responsible for the patients? 
This study will include 20 patients with unilateral blindness due to superficial (defined as 
involving the anterior 200 microns of the corneal stroma on ASOCT imaging) corneal stromal 
scars that are post-bacterial/fungal keratitis or post-traumatic with normal intra-ocular 
structures (clear lens and normal fundus/Bscan) and healthy fellow eye. The following 
exclusion criteria will be applied: 
1. Bilateral corneal disease. 
2. Corneal scars with limbal dysfunction (clinically defined as absent limbal palisades 
or conjunctivalization of the cornea) or ocular surface disease including dry eye disease 
(defined as a Schirmer’s test of less than 10mm at 5 minutes). 
3. Unknown etiology, post-herpetic eye disease or eyes with active intra-ocular 
inflammation. 
4. Children (<18 years of age). 
5. Less than 3 months after documented clinical resolution of acute disease. 
6. Inability/refusal to give written informed consent or to undergo any of the 
anterior segment imaging tests. 
 
• Normal volunteers - From where will they be recruited? How many? 
No normal volunteers are needed for this study. 

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• At the time of their participation in the study, will the subjects be involved in any 
other experimental work? Please give details. 
No. 
• List the exact procedures and how often the subjects will be submitted to such 
procedures, i.e collection of urine and faeces, vene-puncture, intubation, special diet, 
drugs administered with dose excluding radio-isotopes or irradiation. 
At the time of the first procedure 10 ml of blood will be drawn from the patient. After 
the second surgical procedure the patients will undergo slit-lamp photography, anterior 
segment optical coherence tomography imaging (non-contact) and confocal microscopy 
imaging of the operated eye. All these imaging procedures are non-invasive. 
 
• State any potential or known hazards of the procedures listed above. How does the 
investigator intend to overcome these? 
Cultivation of epithelial and stromal cells from limbal biopsies is being performed at our 
stem cell biology laboratory for the past 13 years. These cultivated cells have been used for 
clinical therapy and no hazards have been encountered till date.  
• Give any details of any procedures involving radio-isotopes or irradiation. 
No radio-isotopes will be used. 
• What is the investigator's personal experience with the proposed technique? 
The clinical team involved in this study has been performing ex-vivo cultivated limbal 
stem cell transplantation in human eyes for the past 13 years.  
• How will the informed consent be taken and by whom? 
Written Informed Consent will be taken by the PI and Co-PI’s before the patients are 
enrolled for the study. 
• Please details whether the subjects are to be reimbursed expensed incurred during 
participation in the study and also whether or not it is contemplated giving them a gift 
or token of any sort. If so, please state the exact amount. 
The subjects participating in this study will not be charged for the surgical interventions 
or imaging tests. In the event of any unforeseen adverse event the Institute will provide free 
treatment for the same. 
• Is the protocol, copy of the patient's information and consent form appended with the 
application. 
Yes. 
• Action plan for reporting adverse reaction to Ethics Committee and other participants 
of the study. 
Yes. 
 

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• In the event of any adverse reacting arising directly or indirectly during the course of 
the trial, who will bear the financial responsibility for the on-going care of the patient? 
The L V Prasad Eye Institute will bear the financial responsibility.  
• Any other information that you would like to provide to the Ethics Committee. 
No 
 
 
 
Signatures of the Investigators and Co-investigators.