TITLE: bEYElateral STEM CELL – Regeneration of ocular surface in patients affected by bilateral stem cell deficiency in corneal limbus: comparative efficacy analysis of between buccal mucosa’s stem cell and conjuntiva epithelial stem cell

Veneto Region

TIMEFRAME: October 2013 – October 2015 (2 Years)
THEME: Limbal Stem Cell Deficiency
GRANT AMOUNT: 235.000 €
PARTNERS INVOLVED: “Alto Vicentino” ULSS4 Hospital, ULSS12 Hospital and Azienda Ospedaliera Universitaria Integrata di Verona
P.I. : Dr. Diego PONZIN, MD

SUMMARY:The stem cells of the corneal epithelium are located in the limbus. Pathologies/injuries affecting the limbus lead to limbal stem cell deficiency (LSCD) and eventually vision loss. In the last 10 years, reconstruction of the ocular surface using transplantation of corneal epithelial stem cells (SCs) isolated from limbal biopsies and expanded in vitro has proven to be successful, with 70% of patients showing disappearance of clinical symptoms and restoration of visual acuity. However, in patients with bilateral LSCD (no limbus left on both eyes) transplantation of donor-derived cultured limbal SCs is limited by the side effects of immunosuppressants. The goal of this proposal is to develop new therapies based on transplantation of autologous SCs from alternative sources (oral mucosa and conjunctiva) and to evaluate whether they can reprogram/differentiate into corneal epithelial SCs and generate tissue-engineered cell sheets able to reconstruct the corneal epithelium. Self-renewal, potency, differentiation and proliferation of oral mucosal and conjunctival SCs isolated from biopsies of healthy subjects will be evaluated in vitro and their ability to originate a well characterized corneal epithelium tested on different scaffolds, including fibrin glue, amniotic membrane and humal keratoplasty lenticules. Specific epithelial SC culturing media will be developed in order to respond to the quality standards set by the European Union which has classified SC-based therapies as medicinal products. If results will be successful, we will start recruiting bilateral LSCD patients for a phase I cell therapy clinical trial (following approval by the Ethical Committee and the National Institute of Health). Grafts of autologous cultured SCs will be prepared in the Cell Factory of the Coordinator under GMP conditions. Ten patients with bilateral LSCD will initially be treated (5 transplanted with oral mucosal SCs and 5 with conjunctival SCs) and monitored for at least 1 year.

OBJECTIVES/SCOPE :The goal of this study is therefore to evaluate whether new approaches based on transplantation of autologous stem cells from alternative sources (oral mucosa and conjunctiva) could be a potential therapeutic treatments for patients with bilateral LSCD, thus avoiding the need for immunosuppressive therapy and the risks and costs associated with allograft immunologic rejection.

SUMMARY OF METHODS: The aims of the proposed research are:

(1) to set up in vitro cultures of oral mucosal and (2) of conjunctival stem cells able to reprogram and differentiate into corneal epithelium and to assess both maintenance of self-renewal/proliferation and development of differentiation processes;

(3) to evaluate the ability of oral mucosal & conjunctival stem cells to regenerate a corneal epithelium onto different scaffolds using newly developed cell culturing media and reagents that respond to the standards of safety required for human medicinal products;

(4) to perform a phase I clinical study comparing these 2 strategies by using grafts prepared in a certified Cell Factory with reagents that comply with the guidelines of the European Union for the production of stem cell-based medicinal products (Good Manufacturing Practices, i.e., GMP). 


Transplantation of allogeneic tissues necessitates post-operative long-term systemic immunosuppression, with unfavourable benefit/risk ratios and high costs for the national health system. In addition, even after systemic immunosuppression the long-term success rate (>1 year) ranges from 20 to 60% (cadaveric donors) and from 70 to 80% (living-related donors), with success rates decreasing gradually with time. The possibility to use alternative sources of autologous stem cells that can be reprogrammed to replace LSCs might avoid severe complications due to the immunosuppression regimen. Therapeutic benefits for bilateral LSCD patients could be immediate, particularly for those transplanted with oral mucosal stem cells. Transplantation of autologous oral mucosal grafts directly onto ocular surfaces is currently used for the treatment of corneal ulcers and perforations, lid abnormalities, etc. and is normally accompanied by a reduction in discomfort/foreign body sensation, tearing, photosensitivity and pain. However, these grafts are not useful for improving vision, since they contain opaque sub-epithelial fibrous tissue. In contrast, the transparency of grafts made of tissue-engineered epithelial cells fabricated from oral mucosal epithelial cells would be similar to the transparency of cell sheets originating from LSCs. For all these reasons, transplantation of cultured oral mucosal stem cells would have an immediate triple advantage: (a) to recover a stable and clear corneal epithelium, (b) to reduce pain/discomfort and (c) to allow a recovery of the visual function. 






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