Comparison of retinal pigment epithelial differentiation efficiency from human induced pluripotent stem cells using kit-based and nicotinamide-induced methods

Objective  To compare the differentiation efficiency of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells using the Nuwacell ^® RPE kit and nicotinamide (NIC)-based differentiation methods.

Methods  Normal hiPSC lines and hiPSC lines from peripheral blood of two Bietti crystalline corneoretinal dystrophy (BCD) patients with CYP4V2 gene mutation at passages 16-18, were cultured for 4 days, and were induced into Nuwacell-RPE and NIC-RPE cells using the kit-based differentiation method and the NIC differentiation method, respectively.The percentage of pigmented cells or pigment foci areas formed at 3, 4, and 5 weeks after induction was observed and quantified under a light microscope.The cellular pigmentation level was measured by spectrophotometry.The expression of maturation markers in RPE cells was detected by real-time quantitative PCR and immunofluorescence staining.Cell polarity structures were examined by transmission electron microscopy.The induced RPE cells were co-cultured with porcine photoreceptor outer segments for 48 hours, and the number of rhodopsin (RHO)-labeled POS phagocytosed was quantified by immunofluorescence staining.Transepithelial electrical resistance was measured to evaluate the barrier function of the cells.According to the approval of the Ethics Committee of the First Affiliated Hospital of Army Medical University of China (No.[A]KY2023007), and peripheral blood from two BCD patients with CYP4V2 gene mutation was collected with written informed consent.

Results  The Nuwacell method formed pigmented cells at 3 weeks, with (54.513±5.795)% pigmentation at 5 weeks, while the NIC method formed pigment spots at 4 weeks, with (26.037±8.489)% pigmentation at 5 weeks, with a statistically significant difference ( P<0.005).After 30 days, both methods produced hexagonal RPE structures, with pigmentation levels of (40.060±4.076) and (57.292±2.588)pg/cell, respectively, and the difference was statistically significant ( P<0.001).PCR and immunofluorescence staining showed that increased expression of maturation marker genes RPE65, BEST1, and CRALBP induced by the two methods.NIC-RPE cells phagocytosed more RHO-labeled outer segments than Nuwacell-RPE, showing a statistically significant difference ( t=2.920, P=0.043).After 4 weeks of culture in Transwell chambers, the transepithelial electrical resistance of NIC-RPE was higher than Nuwacell-RPE cells, demonstrating a statistically significant difference ( P<0.05).After continuous passage for 4 generations, Nuwacell-RPE partially lost its original cellular characteristics, including loss of pigmentation, irregular shape and loss of RPE marker expression, while NIC-RPE maintained its original morphologic features.

Conclusions  The Nuwacell kit method has higher differentiation efficiency and RPE cell yield than the NIC-directed differentiation method, and cell maturity, phagocytosis, and barrier functions are inferior to those of the NIC-directed differentiation method.

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