Abstract
Purpose :
We have characterized human trabecular meshwork stem cells (TMSCs) and proved TMSCs can home to the TM for TM regeneration. In this study, we aimed to detect the localization of TMSCs in mice and to determine if endogenous TMSCs can be activated in vivo.
Methods :
Newborn C57BL/6 mice starting at day 3 were injected peritoneally with thymidine analogue brumodeoxyuridine (BrdU) twice a day for three days. After chasing periods of 4-hr, 1-day, 3-day, 1, 2, 3, 4, 8 and 15-week, mice were sacrificed and the anterior portion of the eyes including the TM were dissected and processed for wholemount staining with BrdU and stem cell marker OCT4 antibodies. To examine endogenous TMSC activation, newborn mice were injected with chlorodeoxyuridine (CldU) for 10-week chasing period followed by laser photocoagulation on the TM region. After laser, mice were injected with iododeoxyuridine (IdU) twice for incorporation into new dividing cells. At 4-hr, 24-hr and 1-wk, immunofluorescent staining was performed with anti-CldU, IdU, Histone H3 antibodies on both wholemounts and cryosections. All samples were imaged using a confocal microscopy. Positive stained cells were counted and compared.
Results :
BrdU label-retaining cells were detected in mouse TM at every chasing period. BrdU+ cells were throughout the TM region in short chasing periods before 3-wk. At 4-wk, 8-wk and 15-wk chasing periods, BrdU label-retaining cells were limited at the insert region and most of the BrdU+ cells expressed stem cell marker OCT4. After laser, the numbers of both CldU label-retaining TMSCs and IdU+ dividing cells, as well as double CldU/IdU positive cells (dividing stem cells) were dramatically increased comparing to no-laser controls. Most of incorporated cells were H3+ indicating they were mitotic cells dividing. CldU+ cells were detected in the TM but not limited to the insert region which indicates that CldU retaining cells were migrating out of their niche in response to laser.
Conclusions :
TMSCs in mice are slow-cycling label-retaining cells which are mainly localized at the insert region. After laser photocoagulation, these laser-retaining cells could be activated and migrated into the TM region. Our results indicate that endogenous TMSCs could be activated and it may lead to new treatment targeting for TM regeneration and intraocular pressure reduction for glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.