MESENCHYMAL STEM CELLS FOR CORNEA REGENERATION

TITLE: USE OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS FOR BONE AND CORNEA REGENERATION AND FOR THE TREATMENT OF GVHD AFTER HAEMOPOIETIC STEM CELL ALLOTRANSPLANTATION (ITALY)
SPONSOR:

CariVerona Foundation and Veneto Region

CO-SPONSOR: /
TIMEFRAME:

January 2013 – January 2016 (3 Years)

THEME: Limbal Stem Cell Deficiency
STATUS: ONGOING
GRANT AMOUNT:

1.325.000 €

FBOV ROLE: Partner
PARTNERS INVOLVED: University Hospital of Verona (Italy, LEADER), Vicenza Hospital (Italy)
P.I. : Dr. Giuseppe APRILI, MD; Dr. Mauro KRAMPERA, MD

SUMMARY: The summary will mainly focus on the ocular features.

Corneal epithelial stem cells are localized in the limbus, an anatomical structure that circumscribes the cornea and separates it from the conjunctiva. Limbal stem cell deficiency (LSCD) caused by endogenous diseases or corneal epithelium injuries results in complete destruction of corneal epithelium generating conjunctivalization, chronic inflamation, neovascularization, opacification and continuous ulceration of the cornea, which in turn leads to a chronic pain, photophobia, dry eye and, ultimately, to a visual impairment or blindness. Patients with unilateral affection of LSCD can be treated with autologous transplant of  LSC taken from the healthy eye, but this therapeutic approach is not feasible in the case of bilateral affected patients for whom allogeneic LSC transplantation from cadaveric tissue or a HLA-matched donor is the current therapeutic strategy. Allogeneic transplantation is far from being an optimal treatment since it requires prolonged systemic immunosuppression, leading to side-effects such as disorders of liver and renal function, arterial hypertension, bone marrow depletion and neurotoxicity, which compromise the life quality of patients. Moreover, despite of immunosuppression, the success rate of grafting tends to decrease gradually over time (graft survival rate of 40% at 1 year, and 33% at 2 years).

Bone marrow-derived mesenchymal stem cells (BMMSC) are pluripotent cells distinct of the hematopoietic lineage cells with the capacity to differentiate to osteoblasts, chondrocytes and adipocytes. Also, BMMSC have been shown to transdifferentiate into extramesenchymal lineages like endoderm and ectoderm, a level of plasticity characteristic of embrionic stem cells (ESC). However, unlike ESC, these adult stem cells have not tumorigenic properties and can be extracted from patients by simple bone marrow aspiration and easily expanded in vitro. These characteristics make BMMSC a perfect target for regenerative therapies based on using autologous stem cells in order to avoid problems derived from the allotransplantation-induced immunorejection and the systemic immunosupression treatments.

 

OBJECTIVES/SCOPE : This research is intended to exploit the high plasticity of human BMMSC combined with transdifferentiation mechanisms induced by signalling and growth factors to generate a corneal epithelium in vitro, with the final goal of establishing an alternative source of autologous cells for the treatment of patients affected of bilateral LSCD

 

SUMMARY OF METHODS

To date, several works have described the differentiation of human ESCs and induced pluripotent stem cells to epithelial lineage in vitro, highlighting a key role for the cell signalling factors retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) in the process.

 

The aims of the proposed research are:

  1. Transdifferentiate in vitro human BMMSC from healthy donors into corneal epithelial cells. MSC isolated from human BM will be grown on matrigel to preserve undifferentiated state, and then induced to epithelial transdifferentiation by growing them in 3D spheres-forming culture systems with low glucose and serum free medium containing RA and BMP4. To evaluate mesenchymal-epithelial transition process, increasing expression of  epithelial  markers like p63, CK18, E-cadherin and CK14, as well as decreasing expression of mesenchymal markers like alpha-SMA, vimentin and N-cadherin will be measured by immunofluorescence and quantitative RT-PCR. After induction, co-culture systems with LSCs in keratinocyte growth medium containing epidermal growth factor (EGF) will be established in order to achieve differentiation into corneal keratinocytes. Immunofluorescence and quantitative RT-PCR will be used to detect expression of the corneal differentiation markers CK3/CK12. Then, self-renewal and proliferation capacity of transdifferentiated cells will be measured.  
  2. Generate corneal epithelium in vitro. BMMSC-derived corneal epithelial cells will be growth in vitro onto different carriers/scaffolds appropriate for LSC transplantation like amniotic membrane, fibrin gels and human keratoplasty lenticules. Confluent monolayers will be exposed to air-liquid interface to induce epithelial stratification. Structural integrity and functional differentiation of the epithelium will be assessed by measuring transepithelial electrical resistance and by immunofluorescence and confocal microscopy analysis of epithelial markers like Beta1-integrin, E-Cadherin, tight junction proteins and connexins, as well as, coherent expression of  CK14 and CK15 in basal layer and CK3/CK12 in suprabasal layers.  
  3. Evaluate the regenerative potential of BMMSC-derived corneal epithelial cells in vivo. Carriers/scaffolds containing monolayers of transdifferentiated cells will be grafted on the eyes of animal models of LSCD and long-term graft survival will be evaluated. The capacity of these cells to maintain epithelial stem/progenitor cell character in vivo will be dertermined by the analysis of LSC markers like p63, CK19 and ABCG-2 in the limbal region by immunofluorescence and confocal microscopy and subsequent isolation and clonal cell growth assessment in vitro.

Design a protocol for a phase I clinical trial. If the outcomes of the previous preclinical studies are successful, the final step of the project will be evaluate the efficiency of these cells in patients of bilateral LSCD.

 

EXPECTED RESULTS: This research project will provide an efficient protocol to transdifferentiate adult BMMSC into corneal keratinocytes suitable for autologous transplantation in patients with bilateral LSCD caused by mechanical or chemical injuries, or to be used in ex vivo gene therapy protocols to correct bilateral LSCD associated with congenital disorders like ectrodactyly-ectodermal dysplasia clefting syndrome.

 

PUBLICATIONS:

 

ABSTRACTS/POSTERS TO CONFERENCES:

 

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