Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells

Abstract

Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs).

Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue.

Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue.

Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

FIG. 1.

Human trabecular meshwork (HTM) cells express surface markers consistent with human mesenchymal stem cells (hMSCs). Donor cells extracted from 3 meshworks (HTM 50a, 50b, and 63a; n=3) all expressed positive markers of hMSCs (CD73, CD90, CD105, CD146) and did not express negative markers (CD31, CD34, CD45). This was consistent with the positive control of an adipose-derived hMSC (hMSC; n=1).

FIG. 2.

HTMs and hMSCs have similar expression of self-renewal transcription factors but differ in Dex responsiveness. (A) When compared to hMSCs, HTMs express equivalent or higher levels of the self-renewal/pluripotency genes sox2 and pouf5f1. They additionally express similar levels of notch1, a transmembrane receptor known to be expressed by hMSCs and important in differentiation (n=3; representative results shown). (B) The expression of these proteins is not influenced by 10⁻⁷ M dexamethasone (Dex) treatment when compared to the vehicle control (EtOH). Data mean±standard error of the mean of cells from 3 (hMSC) and 6 (HTM) donors. (C) hMSCs (donors J, D, and H) myoc and angptl7 expression is far lower than HTM cells and lacks the consistent and robust HTM response to Dex. Significance between EtOH/Dex indicated with *, **, ***. Significance between HTM/hMSC indicated with ^###. “n/e” indicates no reliable detectable expression.

FIG. 3.

HTMs express low levels of the OCT4A isoform. When compared to the highly expressing F9 embryonal carcinoma cell line, HTM cells exhibit distinct, albeit limited, immunostaining at the appropriate molecular weight. For clarity of the HTM band, the F9 control was overexposed. Heat shock protein-90 (HSP90) used as a loading control. Blot representative of 4 HTM cultures.

FIG. 4.

HTM cells have a passage-dependent immunomodulatory effect. (A) Log BrdU fluorescence (horizontal axis) and forward scatter (vertical axis) flow cytometry data. T cells in monoculture have limited proliferation without phytohemagglutinin (PHA) and substantial proliferation with PHA. The effect of PHA is inhibited by coculture with hMSCs or HTM cells. (B) Quantification of BrdU-positive cells for the different experimental conditions. HTM cells exhibited a passage-dependent immunomodulatory effect. Color images available online at www.liebertpub.com/jop

FIG. 5.

HTM cells do not differentiate into adipocytes or osteocytes. HTM cells and hMSCs were exposed to adipogenic and osteogenic media for 19 days. Adipogenic and osteogenic differentiation of the hMSCs was indicated by the presence of lipids and alkaline phosphatase, respectively. HTMs did not stain for lipids or alkaline phosphatase with or without differentiation stimulus. Scale bar is 50 μm. Color images available online at www.liebertpub.com/jop

FIG. 6.

Aqueous humor does not differentiate hMSCs into HTM cells. (A) Expression of myoc and angptl7 mRNA remained lower than control HTM cells. Additionally, there was a limited and inconsistent response to 100 nM Dex. (B) Pluripotency factors sox2 and pou5f1 were significantly decreased by culture in 20% bovine aqueous humor (AH) but not 100% AH. Expression of notch1 was unaffected by either media. In all cases, 100 nM Dex treatment reduced the expression of sox2 in control media and 100% AH, and notch1 and pou5f1. Significance between EtOH/Dex indicated with *, **, ***. Significant difference from HTM control (for myoc/angptl7) or from control hMSC (for sox2/pou5f1) indicated with ^###. “n/e” indicates no detectable expression. (C) Alkaline phosphatase staining of hMSCs. Minimal staining is observed in control or 100% AH cultured cells, but prominent staining is observed in 20% AH and osteogenic cultures. Scale bar is 50 μm. Color images available online at www.liebertpub.com/jop

FIG. 7.

HTM cells express α-smooth muscle actin (αSMA). (A) Fresh HTM explant cultures stain strongly positive for αSMA, whereas hMSCs have sparse immunoreactivity. (B) HTM explant cultures have higher mRNA expression than transforming growth factor-beta-treated human corneal fibroblasts (HCF+). Untreated human corneal fibroblasts are used as a negative control (HCF−). Significance between groups indicated with ***. (C) The HTM stains weakly positive for αSMA overall. Ciliary muscle exhibits strong immunoreactivity, which is diminished in the posterior meshwork, but the anterior meshwork (C′) and the insert region (C′′) display individually strongly immunoreactive cells. Scale bars are 50 μm. Color images available online at www.liebertpub.com/jop