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Wei Zhu, Oliver W Gramlich, Budd A Tucker, Markus H Kuehn; iPSC-derived TM cells promote proliferation of primary TM cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4680.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that transplantation of induced pluripotent stem cell-derived trabecular meshwork cells (iPSC-TM) restores normal trabecular meshwork (TM) function in MYOCY437H transgenic mice. The goal of this study was to investigate the interactions between iPSC-TM and primary TM cells (pTM) in vitro and using perfusion cultured human eyes.
50,000 mouse pTM cells were transfected with Ad5RSVmyocilinY437HHisFlag and subsequently either co-cultured with 50,000 purified mouse dsRed+ iPSC-TM either allowing cell-cell contact, separated using transwell inserts, or grown in cell culture media pre-conditioned by iPSC-TM. After 4 days the surviving dsRed-negative cells were counted by flow cytometry. The proliferation rate was analyzed based upon BrdU incorporation within a 2-hour period. Additionally 5 pairs of human donor eyes (83±9.6 yrs old), maintained in a perfusion organ culture system, received transplants of 200,000 human dsRed-positive iPSC-TM. After 2 weeks in culture, TM cells were isolated and counted by flow cytometry and the tissue was evaluated using histochemical and immunohistochemical methods.
After 4 days in culture the number of mouse pTM cells maintained alone increased by 38.7%±6.0 whereas direct co-culture of pTM and iPSC-TM resulted in a 234.4%±68 increase (P=0.0039). The fraction of BrdU positive pTM cells in co-cultures was significant higher than that measured in controls (32.9% vs 21.6%, P=0.03). Significantly heightened proliferation rates were not observed in co-culture experiments when iPSC-TM were maintained in cell culture inserts (19.7%, P=0.16) or in iPSC-TM conditioned media (25.3%, P=0.08). Similar findings were also obtained in human eyes using a perfusion organ culture system. 4 out of 5 donors responded with vigorous TM cell proliferation upon transplantation of iPSC-TM. Although only a small number of surviving iPSC-TM was detected these eyes displayed a 2.1±0.98 fold increase in TM cellularity when compared to the untreated contralateral eye (P=0.035).
These studies demonstrate that iPSC-TM induce a proliferative response in both human and mouse TM cells that requires direct cell-cell contact. Importantly, TM cell proliferation can also be induced in aged human donor eyes, suggesting that this is a general response and not specifically related to myocilin mutation induced TM abnormalities.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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