Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells

Abstract

Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

Keywords: Alkaline phosphatase; Aqueous humor; Mesenchymal stem cells; Myocilin; Osteogenic potential.

Figure 1. ASCs exhibited a time and dose dependent response to aqueous humor

ASCs were cultured in full media (serum supplemented DMEM) in the presence of 5, 10, or 20% AH. (A) Cells were stained for alkaline phosphatase (ALP) activity (red) and with Alcian blue (blue). Some ALP activity was observed in untreated cells (Control), but cells treated with AH exhibit dramatic increase in ALP activity, exemplified by cells cultured for 3 weeks in the presence of 5% AH (3 week - 5% AH). There was apparently less effect of AH on Alcian blue staining. (B) At 2 weeks, ALP activity was increased in a dose dependent manner, with 10% and 20% eliciting a significant response when compared to control cells. (C) At 3 weeks, all doses resulted in a significant increase in ALP activity compared to control. All data were normalized to the response of 3 week treatment with 20% AH. Data are mean ± SEM (n = 3 donors). * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar 50 μm.

Figure 2. Serum was not required for the osteogenic effect of aqueous humor on ASC

ASCs were cultured serum free (un-supplemented DMEM) in the presence of 5, 10, or 20% AH. (A) Cells were stained for ALP activity (red) and with Alcian blue (blue). ALP activity was virtually absent in untreated cells (Control), but cells treated with AH exhibited noticeable ALP activity, exemplified by cells cultured for 3 weeks in the presence of 20% AH (3 week - 20% AH). There was less effect of AH on Alcian blue staining. (B) At 2 weeks, ALP activity was not significantly increased at any dose, although there was an apparent trend towards increase. (C) At 3 weeks, there was a dose response, and cells treated with 20% exhibited a significant increase over control. All data were normalized to the response of 3 week treatment with 20% AH in serum containing media. Note the smaller Y-axis when compared to Figure 1. Data are mean ± SEM (n = 3 donors). * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar 50 μm.

Figure 3. The effect of aqueous humor on the osteogenic potential of ASCs was dependent on thermally labile and charcoal-adsorbable components

ASCs were cultured for 3 weeks in full media (serum supplemented DMEM) in the presence of 5% AH. The AH was either untreated, incubated with dextran-coated charcoal (DCC), or heat treated. (A) Both DCC (5% AH/DCC) and HT (5% AH/HT) treatments reduced ALP activity when compared to untreated AH (5% AH), with HT reducing activity to the level seen in untreated cells (Control). (B) Both DCC and HT significantly reduced the ALP induction caused by AH. The effect of DCC was partial, and was still significantly elevated compared to control cells, while HT resulted in complete loss of AH osteogenic activity. All data normalized to the response of 5% AH case. Data are mean ± SEM (n = 3 donors). * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar 50 μm.

Figure 4. Myocilin partially rescued the loss of activity induced by HT

ASCs were cultured for 3 weeks in full media (serum supplemented DMEM) in the presence of 3 μg/mL myocilin (3 μg/mL myocilin), 5% aqueous humor (5% AH), heat treated 5% AH (5% AH/HT), or myocilin and 5% AH/HT (5% AH/HT + myocilin). (A) Myocilin, both separately and in conjunction with AH/HT, increased ALP activity. (B) Quantification of ALP activity. All data normalized to the response of 5% AH case. Data are mean ± SEM (n = 3 donors). * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar 50 μm.