Abstract
Purpose :
There have been no studies to date of differentiating adult stem cells into retinal ganglion cells (RGCs). The issue with iPSC differentiation is a probability for unlimited proliferation. This has a concern of tumor formation. Our hypothesis was to test if human trabecular meshwork stem cells (TMSCs) from the eye could be induced to RGCs in vitro which provides potential for RGC regeneration.
Methods :
Primary TMSCs were obtained from the de-identified human corneas received from Center of Organ Recovery and Education. TMSCs were characterized for different stem cell markers using flow cytometry and qPCR and used thereafter for RGC differentiation. TMSCs were placed in Neurobasal medium: DMEM (1:1) supplemented with N2B27 and Forskolin (20µM) for 40 days with medium change every third day for induction to differentiate into RGCs. Morphologic changes were evaluated by phase contrast microscopy. Thy1.1 expression was evaluated using flow cytometry. Immunofluorescent staining was performed to detect the expression of BRN3A, BRN3B, and RBPMS which are RGC specific antibodies and cells were photographed using a laser scanning confocal microscope. Statistical analysis was done using a t-test. P<0.05 was considered as statistically significance.
Results :
Isolated TMSCs showed positivity for stem cell markers CD90, CD73, CD105, OCT4, ABCG2, and KLF4 as evaluated by flow cytometry and qPCR. After 40 days of induction, TMSCs differentiated into RGCs with extensive elongated axons and dendrites as evidence from their morphological transformation using light microscopy. Flow cytometry analysis showed that N2B27 + Forskolin induced 2.23% ± 0.79% of Thy1.1+ RGCs while undifferentiated TMSCs had 1.36% ± 0.06% Thy1.1+ cells. The difference was significant (p=0.0068). Fluorescence microscopy images indicated the induced cells in the medium containing N2B27+ Forskolin had positive staining for BRN3A, BRN3B and RBPMS.
Conclusions :
TMSCs were successfully induced to differentiate into RGCs which opens up new avenues using RGCs differentiated from adult stem cells for clinical translational purposes since adult stem cells are safer pertaining to tumor formation.
This is a 2020 ARVO Annual Meeting abstract.