Sudhakar & Sreekanth Ravi Stem Cell Biology Laboratory Champalimaud Translational Centre For Eye Research (C-TRACER)
The stem cell laboratory, set up with the generous support of two brothers - Sudhakar Ravi and Sreekanth Ravi of California, USA - is engaged in frontline research designed to apply knowledge of stem cell biology to the treatment of eye diseases. Stem cells, or undifferentiated cells from a donor eye, are cultured in a special medium to generate corneal cells that can then be transplanted into the eye of a person with corneal blindness. In the area of ocular surface damage, the laboratory has been particularly successful in developing a technique of culturing limbal stem cells ex-vivo on an amniotic membrane substrate and transplanting the resultant corneal epithelium into the patient's eye. The research team is currently exploring various cell therapeutic possibilities for treating corneal surface and stromal damage using cultured limbal stromal stem cells and mesenchymal stem cells; for addressing the problems of corneal endothelial damage, trabecular meshwork dysfunction and retinal dystrophies using different adult stem cells and pluripotent stem cells.
Click here for details about the collaborative effort between Dr Virender Singh Sangwan from the L V Prasad Eye Institute and Professor Sheila MacNeil of Sheffield University
Development of a synthetic bio-degradable cell carrier membrane for the transplantation of cultured cells or freshly excised autologous tissue (limbal segments) for diseases of the cornea Investigators: Virender Singh Sangwan, 1Sheila MacNeil, 1Tony Ryan, 1Frederik Claeyssens, Charanya Ramachandran, Indumathi Mariappan, D Balasubramanian Support: Wellcome Trust, United Kingdom; Department of Biotechnology, India
When the outer epithelial layer of the cornea is damaged by chemical or fire burns, vision is compromised. In some cases, stem cell therapy can be used to generate a functional outer corneal layer to offer significant restoration of vision. The human amniotic membrane is currently the most commonly used substrate for culturing and transplanting limbal stem cells. While this procedure is successful, the study investigates whether it is possible to replace the amniotic membrane, so as to avoid viral contamination, shelf life degradation and other potential risks associated with the use of a biological material. A synthetic biodegradable polymer membrane developed by our collaborators at the University of Sheffield promises to be valuable. Our research thus far shows that the polymer scaffold, similar to the human amniotic membrane, supports sufficient stem cell growth and allows successful transfer of the cultured cells onto the wounded cornea. Following the promising laboratory data and rabbit toxicity results, a pilot clinical trial to test the safety and efficacy of the polymer scaffold has been planned.
Regenerative prostheses as alternative to donor corneas for transplantation to treat blindness Investigators: Virender Singh Sangwan, 2May Griffith, 2Per Fagerholm, 2Jaywant Phopase, Jagadish Reddy, Sayan Basu, Indumathi Mariappan Funding support: Swedish Governmental Agency for Innovation Systems (VINNOVA), Sweden; Department of Biotechnology, India
The objective of this project is to develop and test in the clinic a regenerative prosthesis (combination regeneration promoting implant-prosthetic device) that can potentially eliminate the need for donor corneas and long-term immune suppression for specific corneal transplantation procedures. This corneal implant will be fabricated using human recombinant collagen, with modifications to support their long term stability in vivo.
Exploring the application of pluripotent and adult stem cells in the treatment of retinal and corneal disorders. Investigators: Indumathi Mariappan, Vivek Pravin Dave, Subhadra Jalali, Chitra Kannabiran, Virender Singh Sangwan, Tara Prasad Das, Dorairajan Balasubramanian Funding support: Department of Biotechnology, India
Our group is currently exploring the applications of pluripotent stem cells (PSCs) in the treatment of retinal and corneal disorders. Towards this effort, this project aims to establish efficient protocols for differentiating pluripotent stem cells into retinal and corneal lineages, for the enrichment of differentiated cells into homogenous populations and also to evaluate their suitability in treating disease conditions in pre-clinical animal models.
Creating zebra fish models of retinal dystrophy using genome editing methods. Investigators: Indumathi Mariappan, 3Rakesh Mishra
Funding support: Department of Biotechnology, India
Mutations in several genes involved in phototransduction pathway, vitamin A metabolism, photoreceptor specific transport proteins, transcription and splicing factors are linked to retinal dystrophies. However, the effects of only certain pathogenic mutations are well understood. This study aims to employ genome editing tools to generate near-identical zebra fish models of human gene mutations implicated in disease and aims to understand their effects on retinal development and function.
Proof-of-concept experimentation on gene correction in patient-specific induced pluripotent stem cells by genome editing approach. Investigators: Indumathi Mariappan, Chitra Kannabiran, Dorairajan Balasubramanian
Funding support: Department of Science & Technology, India
This project aims to correct the known genetic mutations in retinal dystrophic patient-specific induced pluripotent stem cells. Mutation correction will be attempted either by delivering a normal copy of the mutated gene, at a safe locus in the genome or by editing the mutation in situ using the latest genome editing tools such as the TALEN and CRISPR systems.
To study the role of myofibroblast/stem cells in corneal wound healing responses: Identifying and characterizing growth factor-receptor systems and herbal/stem cells treatments during development, homeostasis, and corneal wound healing Investigators: Vivek Singh, Virender Singh Sangwan, Sayan Basu
Funding support: Department of Science and Technology, India
Corneal disease is the fourth-leading cause of blindness. According to the World Health Organization, roughly 1.6 million people globally are blind as a result of this disease. The estimated number of people visually impaired in the world is 285 million, 39 million blind and 246 million having low vision; 65 % of people visually impaired and 82% of all blind are 50 years and older; as reported in WHO global data of visual impairments 2010. The current proposal focuses on generating required knowledge base supported with in vivo and in vitro evidences to better understand the complexity associated with wound healing process in cornea as well as limbus and search for better therapeutic modalities and drugs for treatment and disease management associated with Corneal Haze/ limbal wound.
Clinically, a better understanding of the outcomes of corneal surgical procedures and better design of pharmacologic treatments to control anomalous responses such as corneal scaring, inflammatory disorders like diffuse lamellar keratitis, and other wound healing abnormalities would be possible , if there existed a better understanding of 1) cell-cell interactions in the cornea, 2) the mechanisms of action of key modulators of stromal-epithelial interactions in the cornea, 3) the factors regulating immune cell migration into the cornea, and 4) the functions performed by corneal, lacrimal, stem cells and immune cells during the wound healing process. In addition such an understanding would also lead to treatments that increase scaring in the cornea where it is beneficial, for example at the donor-recipient junction in corneal transplantation. It is hypothesised that the human/mouse corneal stromal fibroblast and bone marrow-derived cell/ stem cells interactions augment corneal myofibroblast generation under regulatory control of paracrine and juxtacrine mechanisms.
To study the Biology of Simple limbal epithelial transplantation (SLET), a technique of in-vivo expansion of limbal stem cell for treatment of Limbal stem cell deficiency (LSCD). Investigators: Vivek Singh, Sayan Basu, Virender Singh Sangwan
Funding support: CORE Grant/TKCI/ HERF, Hyderabad
Corneal transparency is a necessary requirement for optimal vision, offering about 70% of the refraction and focusing of incoming light. However, just as the skin does, cornea too reliant on a self-renewal program to preserve its integrity, thus keeping the ocular outer surface stable and functional. Epithelial regeneration capacity of cornea is either regressed or lost in cases of limbal stem cell deficiency (LSCD) which still poses a challenge for visual rehabilitation. It can be unilateral, bilateral, partial or total LSCD. Limbal epithelial transplantation serves as an appropriate redressal for severe ocular surface disorders like burns, chemical injuries or diseases like Steven-Johnson syndrome.
In recent years, cell therapy has been widely accepted as a promising approach in the treatment of various disorders. Novel technique called SLET or simplified technique of limbal transplantation which combines many benefits was first conducted at LVPEI, Hyderabad. This simple technique reduces cost as well as visits for the unilateral LSCD-patients. The procedure has less risk of contamination (as the expansion does not take place in a lab) and is very economical too compared to earlier procedure (Cultivated limbal epithelium transplantation) (Vazirani et al., 2013). Presently, our group as well other groups in the world are also working on the possibilities for developing alternative, structurally simple , synthetic biodegradable scaffold based to replace AM (Deshpande et al., Methods Mol Biol. 2013;1014:179-85). Still, the basic biology behind the success of SLET is not well studied and need to be explored in detail regarding the cellular and molecular changes in animal models after this procedure.
Autologous Ex-vivo Cultivated Limbal Stem Cell Transplantation for Treatment of Superficial Corneal Stromal Scars. Investigators: Sayan Basu, Vivek Singh, Virender Singh Sangwan
Funding support: CORE Grant/ HERF
Pre-clinical work suggests human corneal stromal stem cells can be isolated from clinically replicable limbal biopsies, cultivated in conditions suitable for autologous xeno-free 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 autologous cells to regenerate tissue. We foresee the ability of a clinician to isolate limbal stromal cells from a healthy eye, expand the cells, and, after surgically removing the scar tissue from the wounded eye, apply the patient’s own limbal stem cells to regenerate healthy, transparent tissue.
Dissecting the differential expression of specific stem cell markers and Protein kinase C epsilon (PKCe) in the cases of Ocular Surface Squamous Neoplasm(OSSN)and Squamous Cell Carcinoma(SCC) of conjunctiva and eye lids. Investigators: Dilip Kumar Mishra, Swati Kalki, Milind Naik, Vivek Singh
Funding support: CORE Grant/ HERF
Cancer stem cell markers like ABCG2, P63, K14, and K15 are shown to expressed in basal epithelial cells in the skin and many other organs of various regions but not much has been explored in the cases of OSSN and SCC. Our study will focus to look for these markers expression in the cases of OSSN and SCC. PKCe is also present in the proliferative basal layers and up regulated in the SCC. This study will help us to better understand the basic signaling that leads to carcinoma-in situ and squamous cell carcinoma of conjunctiva and eye lids and also how these markers can effect in their invasive properties.
Generating corneal endothelium from the trabecular meshwork stem cells Investigators: Charanya Ramachandran, D Balasubramanian, Virender S. Sangwan
Funding support: Department of Science and Technology, India
Transparency of the cornea, the first refracting surface of the eye, is crucial for experiencing clear vision. A polygonal mosaic of endothelial cells located on the posterior surface of the cornea maintains corneal transparency by regulating the fluid flow from the anterior chamber into the corneal stroma. An imbalance in the hydration control due to corneal endothelial (CE) dysfunction leads to corneal edema and eventually a severe loss of vision. The CE cells become dysfunctional when their density falls below a critical number of approximately 500-1000cells/mm2 which could result from trauma, surgery, systemic diseases and other endothelial dystrophies (e.g. Fuch’s endothelial dystrophy). Since these cells are essentially non-mitotic in vivo, their loss is irreversible and the only successful treatment option available is to replace the dysfunctional host cornea with a healthy donor cornea. Despite advances in surgical techniques and improved success rates, the paucity in the number of healthy donor corneas continues to pose a challenge for timely restoration of vision in these patients. In India alone around millions are blind due to corneal diseases and corneal endothelial dysfunction is the major causative factor Of the million donor corneas required for treatment only 35,000 healthy donor corneas become available every year. With the large gap in the demand and supply of the tissue, the development of alternative solutions for meeting this demand is critical.
The main aim of this proposal is to identify an alternate source of tissue that could be used to generate CE cells that are viable for transplantation. This is because the CE cells are non-dividing in vivo and has very limited proliferative capacity in vitro. Specifically, this project will determine if cells from the trabecular meshwork (TM) can be induced to take up the corneal endothelial phenotype and if the characteristics of the reprogrammed cells are similar to that of the normal CE cells.
Mesenchymal stem cells for limbal stem cell deficiency and vernal keratoconjunctivitis: Identifying the mechanism of homing and immunomodulation of mesenchymal stem cells from corneal stroma, dental pulp, hair follicle, and umbilical cord. Investigators: Sachin Shukla, Virender Singh Sangwan
Funding support: Department of Science and Technology, India
Limbal stem cells are critical in maintaining the transparency and normal physiology of the cornea by renewing the outermost layer of the cornea - corneal epithelium, at regular interval. Loss of these cells results in partial to complete overlapping of cornea and conjunctiva causing conjunctivalizaton. This leads to a disease called Limbal Stem Cell Deficiency (LSCD) which may cause significant visual loss, corneal injury and blindness. The disease can be caused by thermal or chemical injuries or genetic diseases (e.g., Aniridia).
The goal of the project is to use the autologous adult mesenchymal stem cells (MSCs) for regeneration of the corneal epithelium in bilateral LSCD (both eyes have deficiency of limbal stem cells) patients who do not have any autologous source of healthy limbus available for transplantation. The aim is to understand the mechanism by which the transplanted MSCs reach to the site of injury, regenerate the corneal epithelium and further suppress the elicited immune response. The long term goal is to develop MSC – based cell therapy for treatment of inflammatory and degenerative corneal diseases leading to blindness.
University of Sheffield, UK
Linköping University, Sweden
Centre for Cellular and Molecular Biology, Hyderabad