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David M. Gamm, Jason S. Meyer, Kyle A. Wallace, Amelia D. Verhoeven, De-Ann M. Pillers, Bikash R. Pattnaik; Photoreceptor- and RPE-like Cells Derived from Human Pluripotent Stem Cells Display Characteristic Responses to Small Molecule Stimulation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5994.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the physiological responses of human pluripotent stem cell (hPSC)-derived retinal cell populations following exposure to selected small molecules.
Isolated optic vesicle(OV)-stage neurospheres derived from hPSCs were differentiated to photoreceptor(PR)- and/or RPE-like cells using an established protocol. Current responses of individual PR-like cells to applied voltage were recorded in the presence or absence of agonists and antagonists using standard whole-cell and perforated patch-clamp configurations. After recording, cells were backfilled with sulphorhodamine to facilitate identification. To monitor [Ca2+]i changes in response to ATP, hPSC-RPE cells were first loaded with Fura2AM. Thereafter, fluorescence imaging was performed to obtain time-dependent, agonist-induced [Ca2+]i measurements.
Differentiating OV neurospheres expressed appropriate gene and/or protein markers, including those involved in phototransduction. PR-like cells demonstrated an outward current at depolarizing voltages between -50 and +60 mV (holding potential = -70 mV) that was blocked by TEA. These PR-like cells possessed a resting membrane potential of -44±4 mV and a current density of 34±7.5 pA/pF at +40 mV. Upon treatment with membrane-permeable Br-cGMP, PR-like cells underwent depolarization. hPSC-RPE cells also responded to small molecule stimulation, consistently showing a transient increase in [Ca2+]i after exposure to ATP.
hPSC-derived, PR- and RPE-like cells displayed important functional properties in vitro. Of particular interest, PR-like cells depolarized in the presence of the phototransduction second messenger cGMP, and RPE cells demonstrated an increase in [Ca2+]i in response to exogenous ATP, a molecule postulated to govern the RPE light response. The capacity of hPSC-derived retinal progeny to respond to physiological stimuli in vitro extends their utility as tools for basic science and clinical research.
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