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D. Choudhary; Regulation of Glaucoma-Associated and Trabecular Meshwork-Marker Genes Expression During Human Embryonic Stem Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):728.
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© ARVO (1962-2015); The Authors (2016-present)
Embryonic stem cells (ESCs) have the unique ability of differentiating to multiple cellular types and are a good model system to delineate the molecular changes in early ontogeny by recapitulating the embryonic developmental program under in vitro conditions. The purpose of this study is to characterize the developmental expression pattern of the glaucoma-associated [cytochrome P450 1B1 (CYP1B1), optineurin (OPTN), WDR-36] and TM-marker genes [matrix gla protein (MGP), human cartilage glycoprotein 39 (GP-39) and myocilin (MYOC)] during differentiation of human ESCs.
The hESCs were propagated on irradiated MEFs. Murine stromal OP9 cells were co-cultured with hESCs in differentiation medium (α-MEM, 10% FBS, monothioglycerol). Half of the medium is replaced at day 4, 6, 8 and 10. For EB formation, hESC colonies were passaged to non-tissue culture treated flasks in a suspension medium without FGF2. RNA isolation, cDNA synthesis and gene-specific PCR reactions were performed using standard protocols.
We investigated the expression of the TM-marker genes and glaucoma-associated genes in pluripotent H9 hESC line and during different time points of differentiation in three basic approaches involving spontaneous differentiation of ESCs monolayers, H9/OP9 stromal co-culture and embryoid body (EB) formation. MYOC gene is a common gene to be associated with glaucoma and considered as TM-marker. Undifferentiated hESCs did not show transcripts for the selected TM markers (GP-39, MGP, MYOC) but we did observe the expression of glaucoma-related genes (CYP1B1, OPTN, WDR36). We detected appearance of GP-39 at day 4 and MGP at day 6 which again showed an increasing level of their transcripts with increase in co-culture period. One interesting finding is that the neuroectoderm marker PAX6 and glaucoma related genes (CYP1B1, WDR36) were observed to increase during differentiation induced through the EB formation process as compared to the stromal co-culture differentiation protocol. We also observed a decreasing trend of pluripotent marker genes, OCT4B and SOX2 with increasing time of hESC/OP9 co-culture.
The findings implicate the role of these genes in the embryonic and fetal ontogeny.
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