Implementation of a Protocol for the Transdifferentiation of Buccal Mucosal Epithelium Into Corneal Epithelium

1. Recall on Corneal Anatomy and Histology The cornea is an essential ocular component for vision: often referred to as the first "transparent porthole" at the front of the eye. It is the first structure that light encounters upon entering the eye. Its primary role is to converge incident light rays, which then proceed towards the lens before reaching the retina and initiating the visual cascade. The cornea is an avascular and transparent tissue composed of five layers: corneal epithelium, Bowman's layer, corneal stroma, Descemet's membrane, and corneal endothelium.

• The corneal epithelium is the outermost layer of the cornea, consisting of corneal epithelial cells, accounting for about 10% of the entire cornea. This layer constantly regenerates, with complete renewal occurring every seven days. Scratches on this layer generally heal well.
• The Bowman's layer helps bind the corneal epithelium and stroma together. This layer cannot regenerate once damaged, potentially leading to permanent scars that can affect vision.
• The corneal stroma represents about 90% of the cornea and is composed of collagen and keratocytes.
• Descemet's membrane is made up of collagen and separates the corneal stroma from the endothelium. This layer gradually thickens with age.
• The corneal endothelium is the innermost layer and aids in transporting fluid from other corneal layers. Damage to this layer can compromise this process and negatively impact a person's vision.

1. Renewal and Healing Process of Corneal Epithelium Corneal epithelial cells regenerate within 3 to 10 days through constant renewal from limbal stem cells located at the limbus (transition zone between the cornea and sclera) in the basal cell layer, within pigmented crypts called Vogt's palisades. Deficiency In Limbal Stem Cells (LSCD) is characterized by a loss or impairment of limbal stem cells crucial for corneal epithelial repopulation and limbal barrier function. The etiologies of LSCD can be genetic (PAX6), secondary to chronic inflammation (e.g., severe allergic keratoconjunctivitis or ocular rosacea), acquired through infection such as herpetic keratitis, or secondary to a blistering disease such as Steven-Johnson syndrome or Lyell syndrome. LSCD can also be acquired through trauma from chemical or thermal burns. Etiologies can also be idiopathic. When these stem cells are lost, the corneal epithelium is unable to repair and renew itself. This leads to epithelial degradation, persistent epithelial defects, conjunctivalization, corneal neovascularization, corneal scars, and chronic inflammation. These factors contribute to photophobia, loss of corneal clarity, vision loss, and chronic pain. This makes corneal transplantation impossible as it would be rejected and/or exhibit poor healing. Current solutions for addressing this cause of corneal blindness are limited. Techniques include autologous limbal grafts from the patient's contralateral eye, but this may pose risk and is not possible when the pathology is bilateral. Allogeneic grafts are highly risky for rejection and require systemic immunosuppressive treatment.
2. Mucous Membrane Grafts in Periorbital Reconstruction

Oral mucosa grafting is a well-established surgical technique for treating conjunctival deficiencies and scars. Oral mucosa possesses biological properties similar to conjunctiva, both being composed of one or more layers of epithelial cells covering a loose connective tissue layer. It can be harvested repeatedly, with the donor site easily accessible and widely available. Donor site morbidity is low, and surgery is generally well-tolerated by patients. Moreover, the surgical technique is relatively simple, making oral mucosa an ideal candidate for replacing conjunctival anomalies.

Oral mucosa grafts have been used not only for treating contracted sockets in anophthalmic patients and reconstructing the ocular surface and fornix in patients after tumor resection but also for treating refractive pterygium and in patients with pemphigoid or ocular symblepharon. It is also the most commonly used tissue in treating post-surgical complications, including conjunctival deficiencies after glaucoma, retinal surgery, corneal melts related to keratoprosthesis, and covering the dacryocystorhinostomy tract. Additionally, oral mucosa has been used to repair intractable sclerocorneal melts caused by severe chemical burns.

Some teams have proposed using buccal mucosa cells and transdifferentiating them into limbal stem cells. The oral cavity is easily accessible, and stem cells can be isolated from the mucosa in a minimally invasive and low-risk manner for the patient. These cells have a high differentiation potential and express markers of embryonic stem cells; Tra2-49, Tra2-54, SSEA4, Oct4, Sox2, and Nanog, as well as the neural crest marker Nestin. However, human trials are still in the preliminary stage, and no therapy has been validated to date.

In this proof-of-concept study, the aim is to develop a protocol for differentiating oral mucosa cells into limbal stem cells for use in a future clinical trial with patients.