Moving with the Times: Migration of Cultured Keratocytes and Keratocyte Progenitor Cells and the Potential for Cell Based Corneal Transplantation

James McKelvie

Purpose: To characterise the mechanisms initiating cell migration and the subsequent gene expression and division of cultured keratocytes and keratocyte progenitor cell (KPC) spheres.

Methods: Human corneal stromal tissue was enzymatically digested to isolate stromal cells which were subsequently cultured using a sphere forming assay and labelled using fluorescent cytoplasmic Qdot nanocrystals. Labelled spheres were investigated using confocal and fluorescent microscopy. Spheres were placed on collagen coated coverslips and observed with fluorescent time-lapse photography to monitor cell migration. Migrating cells were incubated with Click-iT EdU to confirm cell division. Gene and protein expression of migrating cells, cultured spheres, and freshly digested non-cultured stromal cells were compared using TaqMan quantitative gene array cards and immunohistochemistry.

Results: Digested stroma yielded 7x105 cells/gm. Isolated stromal cells efficiently endocytosed Qdot nanocrystals and were visible with fluorescent and confocal microscopy. Cultured cells formed spheres containing several different coloured Qdots indicating that spheres primarily form from aggregation rather than division of cultured cells. Collagen matrix added to the culture media triggered radial migration away from spheres at speeds of up to 16um/h with concomitant cell division. Immunohistochemistry of cultured spheres demonstrated extracellular matrix deposition including collagen subtypes in similar proportions to that seen in corneal stroma.

Conclusions: It is possible to isolate and culture KPC from human corneal stroma. Cultured cells can be stimulated to migrate and divide with the addition of collagen. These results imply that using cell-based transplants, in the form of cultured spheres, may be possible to treat corneal dystrophies such as keratoconus.