Seeing Your Way Clear: Corneal Stem Cells from Bench to Bedside

Welcome to the first Bitesize Bio article focused on the cornea. As you read this you are peering through at least one cornea—a thin layer of cells on the surface of the eye. The cornea is the eye’s first line of defense against harsh environmental assaults, such as dust, infectious microbes and errant mascara, all while maintaining optical clarity.

In addition to its role as a physical barrier, the cornea has the ability to heal itself after minor injuries. This healing ability is mediated by the limbal stem cells in the basal epithelial layer of the corneoscleral limbus. The corneoscleral limbus exists as a border between the cornea and the white of the eye (the sclera), and is shown in red in Figure 1 below.

Figure 1: Diagrammatic depiction of the human cornea.

The Cornea’s Remarkable Repair Ability

When you think about it, the cornea has a lot of repairing to do! We blink our eyes an average of 15–20 times per minute, clearing away debris and the occasional eyelash that can lead to microscopic scratches and loss of corneal epithelial cells at the eye surface. Limbal stem cells divide throughout an individual’s lifetime, constantly replacing cells that are sloughed off. Corneal cell loss and replacement co-occur in a happy equilibrium on the corneal surface, unless there is major damage due to ocular trauma or disease.

Limbal Stem Cell Insufficiency (LSCD)

Severe eye trauma, degenerative diseases, genetic disorders and excessive contact lens usage can lead to permanent damage, not only to the cornea itself, but also to the resident stem cells that could potentially repair the damage, resulting in limbal stem cell insufficiency (LSCD). LSCD can lead to decreased corneal transparency, chronic pain, light sensitivity and visual impairment.

Stem Cell Replacement Therapy

Stem cell replacement therapy is the only treatment option that can restore a more normal equilibrium. Healing grafts of limbal stem cells can be derived from a small amount of tissue harvested from i) a healthy companion eye (autologous transplant), ii) a healthy eye of a relative or iii) a cadaver. Autologous limbal stem cell grafts offer the best chance for permanent recovery, with a success rate exceeding 80%.¹ The first limbal cell transplantation procedure emerged in the 1960s and nowadays a number of transplantation procedures exist, benefitting thousands of people every single year!

Isolation of Corneal Limbal Stem Cells

Corneal limbal stem cells continue to be studied in vitro for more than just graft optimization experiments. Isolation of corneal limbal stem cells (from human donor eyes or animal models) can be carried out as follows:

1. Careful dissection of corneoscleral rims.²
2. Dissected tissues are briefly rinsed with phosphate buffer, containing antibiotics to prevent bacterial growth.
3. Tissues are either dissociated into a single cell suspension by incubation in 0.05% trypsin for 20 minutes, or grown as organotypic cultures without dissociation, thus maintaining several structural and organizational aspects of the original tissue.
4. Limbal stem cells are identified in culture as Hoechst dye-excluding cells and the expression of specific stem cell markers, such as p63, ABCG2, Vimentin and Cytokeratin K19.

Culture of Limbal Stem Cells – Some Useful Tips!

• Limbal stem cells grow well in keratinocyte serum-free medium (KSFM) or DMEM supplemented with 10% fetal calf serum.
• Be aware that some culture protocols call for a feeder layer of denuded amniotic membrane, lens capsule, or inactivated fibroblasts, while other protocols are feeder-free.
• As with other stem cell types, limbal stem cells undergo self-renewal and continue to proliferate in culture, albeit slowly. Bear in mind that preservation of the stem cell phenotype can be a challenge.
  • For this reason, care must be taken with long-term cultures, since spontaneous differentiation can result in mature corneal epithelial cells that express differentiated markers such as Cytokeratin K3 and Connexin 43. It is therefore wise to measure expression of these mature markers throughout the duration of limbal stem cell culture as a way to monitor the rate of differentiation over time.

With good protocols and careful attention to detail, undifferentiated limbal stem cells can be maintained for numerous downstream in vitro and in vivo experiments, such as flow cytometry, PCR, immunocytochemistry and experimental grafting.

So far, the corneal limbal stem cell is a success story in stem cell replacement therapy, and who knows what advances await us in the future.

References:

1. Vazirani J, et al. (2016) Autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency: multicentre results. Br J Ophthalmol.  100(10):1416–20.

2. Li DQ, Wang Z, Yoon KC, Bian F. (2014) Characterization, isolation, expansion and clinical therapy of human corneal epithelial stem/progenitor cells. J Stem Cells. 9(2):79–91.

3. Graziella P, De Luca M. (2014) Eyes on the Prize: Limbal Stem Cells and Corneal Restoration. Cell Stem Cell 15:121–2.

4. Aaron WJ, Sonia N. (2014) Concise Review: Identifying Limbal Stem Cells: Classical Concepts and New Challenges. Stem Cells Transl Med. 3(3): 318–22.