Hyaluronan Supports Corneal Limbal Stem Cells

Substantial research has shown that the corneal limbus, between the cornea and the conjunctiva, contains limbal stem cells (LSCs), which help maintain corneal homeostasis and regenerate the corneal epithelium after injury. Previous work by our lab has shown that hyaluronan (HA) is a major component of the LSC niche, it helps maintain the LSC phenotype in vivo. This study aimed to investigate whether HA can support LSCs ex vivo. Single LSC suspensions were obtained from human corneas (29-65 years and less than 72 hours post-mortem) by sequential dispase and trypsin digestion. Isolated LSCs were seeded on collagen IV (ColIV), poly-L-lysine (PLL) or PLL/hyaluronan (HA) coated dishes in the presence or not of 3T3 feeder cells. Proliferation, cell size and cell roundness were analyzed at each passage, and, colony formation capabilities (CFA) and expression of LSC markers was analyzed at the end of passage 3. LSCs cultured on PLLHA presented increased CFA capabilities and increased number of cells expressing LSC markers. LSCs cultured on PLLHA had a circularity mean average of 0.8125 for Passage 0, 0.7857 for passage 2, and 0.8039 for passage 3. This shows that LSCs cultured on PLLHA presented significantly increased cell roundness, as these values were close to 1, or perfect cell roundness. The average mean area indicates the average size of one cell. For passage 3, PLLHA had the greatest mean area with a value of 1584.31um2. Thus, this study indicates HA could be used to support the ex vivo expansion of LSCs for LSC transplantation.