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Eric Snider, Richard Taylor Vannatta, W Daniel Stamer, C Ross Ethier; Co-Culture Stimulated Differentiation of Mesenchymal Stem Cells to Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5327.
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© ARVO (1962-2015); The Authors (2016-present)
Due to reduced trabecular meshwork (TM) cellularity in glaucoma, stem cells offer potential therapeutic benefits if properly differentiated and engrafted into TM. Towards this goal, Zhou et al. (ARVO, 2015) studied differentiation of adult adipose-derived mesenchymal stem cells (adMSCs) to a TM lineage by co-culture. Here we further characterize adMSC to TM differentiation by co-culture using genetic, protein-level and functional assays.
Human adMSCs (Lonza; n=2 lines) and primary human TM cells (Stamer lab; n = 3 lines) were examined. We used 3 co-culture approaches with decreasing degrees of adMSC-TM cell interaction: direct co-culture (DCC) after tagging cells to allow later sorting, Transwell co-culture (TWCC), and media swap co-culture (MSCC). Pure TM and adMSC cultures were positive and negative controls. After 1, 2 or 3 wks of co-culture, cells were characterized 3 ways: (i) qRT-PCR; (ii) myocilin induction after 100 nM Dexamethasone (DEX) for 1 wk, assessed by labelling/flow cytometry; and (iii) cell contraction, assayed by area change of collagen I gels 24 hr after cell loading.
adMSCs in DCC showed increased/decreased levels of characteristic TM/MSC message by qRT-PCR (Fig 1A). Similarly, DEX-treated adMSCs from DCC showed increased myocilin expression vs. control MSCs, although myocilin levels were less than in primary TM cultures (positive controls, Fig 1B). By qRT-PCR, adMSCs from TWCC and MSCC showed RNA differences vs. control MSCs, with MSCC exhibiting more TM-like characteristics than TWCC (Fig 2A). However, adMSCs from TWCC were less contractile (similar to TM cells) while adMSCs from MSCC were similar to MSCs (Fig 2B).
Three co-culture paradigms show that adMSCs can differentiate towards a TM-like phenotype, in agreement with Zhou et al, with differentiation extent depending on assay technique. Further optimization of co-culture conditions is needed to increase differentiation towards a full TM phenotype.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Figure 1: DCC Results. (A) qRT-PCR for Muc 1 (MSC marker), MYOC, and tPA (TM markers). mRNA levels are: (i) normalized to geometric mean of 3 reference genes (YWHAZ, TATA BP, GUSB) and (ii) to untreated MSCs. (B) Flow cytometry for myocilin levels in wk 3 samples after DEX.
Figure 2: MSCC and TWCC Results. (A) qRT-PCR (B) Gel contractility for week 2 & 3 samples.
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