A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells

Abstract

Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro.Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded.Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC).Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.

KIT ligand produced by limbal niche cells under control of SOX10 maintains limbal epithelial stem cell survival by activating the KIT/AKT signalling pathway

“…KIT signalling is required for glycogen metabolism of the corneal epithelium, 54 and loss of either KITL or KIT significantly impairs corneal wound healing 55 . Interestingly, KIT is not only expressed in LESCs but also in photoreceptors, lacrimal canaliculus epithelial stem cells and eye wall cells 56‐58 . These cells are all essential for sustaining the physiological functions of the eye and are located in close proximity to SOX10‐positive NCCs or their derivatives 24 .…”

“…55 Interestingly, KIT is not only expressed in LESCs but also in photoreceptors, lacrimal canaliculus epithelial stem cells and eye wall cells. [56][57][58] These cells are all essential for sustaining the physiological functions of the eye and are located in close proximity to SOX10-positive NCCs or their derivatives. 24 Hence, the SOX10-KIT-AKT axis may have a role in eye development and function beyond its role in maintaining LESCs that we demonstrated here.…”