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A. Y. Rose, Y. Chen, T. Acott, M. Kelley; Changes in Protein Expression by Trabecular Meshwork (TM) Cells and Human Mesenchymal Stem Cells (hMSC) Under Different Culture Conditions. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2065. doi: https://doi.org/.
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The TM cell population in the outflow system is reduced with increasing age and with primary open-angle glaucoma. This may contribute to intraocular pressure elevation. Previous studies have shown that hMSC can differentiate into a variety of different cell types. Several factors play a role in hMSC differentiation including growth factors, cell-cell interactions, extracellular matrix (ECM) and cell density. In vivo, TM cells are nourished by aqueous humor (AH). To determine if hMSC can be induced to become more like TM cells for future cell replacement therapy, we investigated several culture conditions, including culturing them in AH. We also studied possible TM markers to monitor these changes.
HMSC, porcine (PTM) and human TM (HTM) cells were used in early passages. HTM and PTM were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) or 50% AH. Control hMSC were grown in hMSC basal media with growth supplements. HMSC were stimulated with adipogenic induction media as a positive control. To investigate the differentiation potential, hMSC were cultured with 50% AH and with TM cells in different proportions on ECM produced by TM cells. Media and cell extracts were analyzed by Western immunoblotting with CD105, CD44, CD10, ABC transporter G2 (ABCG2), chitinase-3-like 1 (YKL-40), human milk fat globulin (HMFG), and optineurin antibodies. Confocal microscopy was used to assess morphology and gene expression. To distinguish between hMSC and TM in mixed cultures, hMSC were infected with a retrovirus expressing green fluorescent protein.
The protein profiles and morphologies of TM and hMSC cells were changed when cells were grown in 50% AH, as well as when the two different cell types were grown together. CD105 (one of the MSC markers) was highly expressed in hMSC cultured in hMSC media, but the expression level decreased dramatically when cells were cultured with 50%AH, as well as in the positive control grown with adipogenic media. ABCG2, optineurin and CD44 were expressed by both TM and hMSC cells. YKL-40, HMFG (putative TM markers) were expressed in TM cells, but also were expressed by other eye tissues. The production of CD10 increased when hMSC were cultured with 50% AH.
Aqueous humor components are partly responsible for the morphological and molecular changes in TM and hMSC. HMSC showed changes in protein profile suggesting that differentiation occurs when cells were cultured in AH or with TM cells, although complete differentiation has not yet been achieved.
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