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Eric Snider, Christopher Pride, Akash Patil, W Daniel Stamer, C Ross Ethier; Characterization of Mesenchymal Stem Cells vs. Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2256.
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© ARVO (1962-2015); The Authors (2016-present)
Due to reduced trabecular meshwork (TM) cellularity in glaucoma, meshwork repair with suitably differentiated stem cells has potential. This requires characterization methods to detect differences between TM and stem cells. The gold standard for identifying TM cells is dexamethasone (dex) induction of myocilin (MYOC); however, to assess differentiation, additional assessments of function and genetic profile are desirable. Here we evaluate multiple techniques to assess differences between mesenchymal stem cells (MSCs) and TM cells.
Human adipose-derived MSCs (Lonza; n=3 strains) and primary human TM cells (Stamer lab; n = 4 strains) were used. Three approaches assessed MSC and TM differences. First, qRT-PCR was performed on RNA isolated from cell lysates. Second, induction of myocilin expression after 500 nM Dex treatment for 1 week was assessed using western blots. Finally, cell contractility was determined by measuring area vs. time in free-floating cell laden collagen I gels.
Differentially expressed mRNAs between MSCs and TM cells were observed (subset shown in Figure 1 for 4 different TM strains where 2 strains with more juxtacanalicular [JCT] cells are noted), with MGP, MYOC and Plas-Act message levels higher in most TM strains. In addition, JCT/TM strains differentially expressed lower CHI3l1 levels compared to TM-only strains. Dex led to c. 20-fold higher MYOC protein expression in TM cells vs. MSCs. TM cells were found to be less contractile than MSCs over 7 - 10 day time scales.
qRT-PCR identified a number of markers upregulated in TM cells vs. MSCs. TM cell and MSC strains were found to be heterogeneous in their expression profile, and subsequent studies should use multiple cell strains to account for cell variability. Dex induction of MYOC was confirmed as a robust but not rapid characterization approach. Differences were also noted in contractile properties of the cell types, with MSCs more contractile than TM cells. Next steps will expose MSCs to relevant biophysical stimuli to establish a differentiation protocol to a TM lineage.
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