Stem-like Cells in Serum Free In-vitro Cultures of Human Lacrimal Gland

Abstract

Purpose: Tear film deficiency due to lacrimal gland dysfunction or damage is an important cause of ocular morbidity. Restoration of gland function by transplantation of autologous ex-vivo expanded stem cells located in the lacrimal gland is one of the options that could relieve this problem. We have previously reported the presence of stem-like and functionally competent differentiated cells in in-vitro cultures of human lacrimal gland. The present study focuses on the formation of ‘lacrispheres’ under serum-free condition and the expression of stemness in them.

Methods: Fresh human lacrimal gland tissues (n=7) from patients undergoing exenteration were harvested for cultures after IRB approval. The gland was processed by enzymatic digestion using a cocktail of collagenase and hyaluronidase. The isolated cells were plated on ultralow attachment plates as group of two-three cells and fed with HepatoStim supplemented with epidermal growth factor, fibroblast growth factor and N2. The spheres were pulse-labeled with BrDU, analyzed for the expression of CD117 by immunocytochemistry and their colony forming efficiency was assessed on Matrigel.

Results: Serum free cultures demonstrate spheres from human lacrimal gland in-vitro within 2-3 days of plating. These spheres grow in size over 3-14 days and can be serially passaged to generate secondary spheres. Anti-BrDU labeling of these spheres indicate the presence of 3.8% of high intensity cells at the periphery and about 3% dull intensity cells at the center. These also show positive labeling for CD117 and formation of clones in Matrigel (CFU 3.1%) indicating the presence of stemness.

Conclusions: This is the first report on the generation of ‘lacrisphers’ in human lacrimal gland cultures. It strengthens our initial reports that human lacrimal gland has a storehouse of stem cells that can be maintained in-vitro and could possibly serve as potential source of cell therapy for the regeneration of the functionally compromised gland.

Keywords: 576 lacrimal gland • 486 cornea: tears/tear film/dry eye • 721 stem cells