BIOENGINEERED CORNEAS

Corneal stromal stem cells can now be isolated reliably from long-term stored organ culture corneas, opening up the possibility to develop cell- and tissue-based therapies for a wide range of corneal diseases, according to Alvena Kureshi PhD.

“We can now differentiate these corneal stromal stem cells into keratocytes which is a useful cell population for bioengineering a corneal stroma or perhaps a cell therapy for corneal scarring,” Dr Kureshi, of University College London, told delegates attending the 5th EuCornea Congress in London.

Corneal blindness remains a leading cause of treatable vision loss worldwide, noted Dr Kureshi, with widespread donor shortages and problems with immune rejection spurring major research efforts to find viable alternatives.

“Currently the most common approach to restoring vision in scarred corneas is to surgically replace the stromal (stroma??) with allogeneic donor cornea tissue. However, there is a decreasing supply of donor corneas worldwide and this is expected to further reduce due to recent advances in corrective eye surgery, which unfortunately renders corneas unsuitable for transplantation,” she said.

Dr Kureshi's research, carried out in collaboration with Prof Jim Funderburgh from the University of Pittsburgh, focuses on using stem cell therapy technologies that are capable of repairing or replacing the scarred corneal stroma. “The key to this is to use cells that are native to the corneal stroma, so we really wanted to find out which would be the ideal cells to use for this purpose,” she added.

Progenitor cells

Stromal stem cells have been identified as the progenitor cells of keratocytes and also support human limbal epithelial stem cells (LESCs), essential for the maintenance of the ocular surface, said Dr Kureshi. “Keratocytes are the main population of cells responsible for synthesising and maintaining the organised ultrastructure of the stroma which is key to its transparency,” she said.

These stromal stem cells hold great therapeutic potential, said Dr Kureshi. “Unlike keratocytes which can be difficult to culture in vitro as they quickly become fibroblasts when you try to expand them, corneal stromal stem cells replicate without loss of differentiation potential. This means that we would potentially be able to culture these cells in large quantities and then differentiate them into keratocytes which would be a useful cell population for bioengineering the corneal stroma,” she said.

The cells could also be potentially used as direct cell-based therapy for corneal scarring, said Dr Kureshi.

“Recent studies using a mouse scar model have shown that these cells have the ability to restore collagen fibril organisation and transparency when injected into the corneal stroma of lumican-deficient mice without immune rejection,” she added.

After learning to isolate and culture the corneal stromal stem cells, Dr Kureshi established that the cells could also potentially survive long-term storage conditions and be successfully differentiated into keratocytes.

For the delivery system, Dr Kureshi proposed utilising a refined system of compressed collagen gels fabricated by a process known as Real Architecture for 3D Tissues (RAFT, TAP Biosystems). To produce 3D cell cultures, a mix of collagen, cells and other reagents is pipetted into the wells of a plate and incubated for 15 minutes to form a collagen hydrogel. The RAFT plate, with its set of biocompatible absorbers, is then placed on top of the gels and over a 15-minute period a controlled amount of liquid is removed from the hydrogel leaving cells “encapsulated” in collagen and evenly distributed in the culture.

Dr Kureshi noted that while the initial RAFT process was quite inconsistent and not very reproducible, collaboration with TAP Biosystems has resulted in a modified approach to make the well plate carrier system more robust, more reproducible and, critically, more compatible with good manufacturing practice, she said.

“We have further evolved the RAFT system for increased clinical applicability and it is now commercially available as a kit with type I collagen and neutralising solutions. The initial paper plungers have now been replaced with hydrophilic porous absorbers which have much less particle shedding and hence are much more appropriate for use in an environment such as a clean room for eventual transplantation to a patient,” she concluded.

Alvena Kureshi: a.kureshi@ucl.ac.uk