Using our recently optimized protocol for differentiating hESC,
22 we generated PRPs expressing the Crx marker, with a relatively high yield (up to 95%) and studied their electrophysiologic maturation throughout the differentiation process up to 90 days. Interestingly, our electrophysiologic investigation revealed that the outward potassium current is observed at an early stage, with recordings from ES cells demonstrating clear outward K
+ currents under the voltage-clamp protocol used for this work. The percentage of cells presenting this current does not increase with maturation, with almost all investigated cells exhibiting this current. This outward current was blocked by TEA and is thus presumably mediated by delayed rectifier potassium channels, similar to Meyer et al.,
14 who reported on outward currents in photoreceptor-like cells derived from optic vesicles, with comparable measured currents (around 400 pA to 450 pA at 40 mV). The delayed rectifier currents play a major role in limiting membrane excitability
30 and are facilitated by a family of potassium channels Kv2,
31 which are modified (suppressed) by the KCNV2 (also known as KV11.1 or Kv8.2 subunit).
24–26 The ratio between the expression levels of KCNB1 (Kv2.1) and KCNV2 (Kv8.2 subunit) determines the outward potassium current in the photoreceptors
26 (with several mutations in the KCNV2 gene
32,33 being associated with cone degenerations). Thus, the gradual increase in KCNB1, accompanied by the constant KCNv2 expression observed in our developing PRPs, is in line with the gradual increase in the measured outward current. Our findings are further supported by Homma et al.,
18 who found a gradual increase in various subunits of the K
+ channel during the postnatal retinal development.