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Fig. 6 | Stem Cell Research & Therapy

Fig. 6

From: Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

Fig. 6

Human PSC-RPE and hPSC-LESCs maintained their cellular phenotypes after cryopreservation. a Human ESC1-RPE were cryopreserved for 52 days (–ind.) or for 281 days (+ind.) and thawed to 1.8 μg/cm2 LN-521 + 10 μg/cm2 col IV combination matrix. After 74 days (–ind.) and 71 days (+ind.) of post-thaw culture on inserts the cells showed (a) mean TEER values of 217 and 151 Ω*cm2 (n = 4 inserts and 6 inserts), (b) expression and localization of zonula occludens-1 (ZO-1) and Claudin-19 (Cl-19) to tight junctions, expression of the visual cycle protein cellular retinaldehyde binding prot (CRALBP), and apical expression of Na+K+ATPase and tyrosine-protein kinase Mer (MERTK); scale bars = 20 μm. c Human ESC1-LESCs, 4 days after recovery from cryostocks (103 days frozen, day 28 of differentiation in total) maintained morphology and expression of LESC markers similar to fresh cells; scale bars = 100 μm. A similar morphology and expression pattern was achieved after replating for expansion (day 34 in total). d Quantification of p40 and PAX6 expression for both hESC-LESCs (hESC1) and hiPSC-LESCs (hiPSC2) confirmed that the high LESC marker expression was retained during subsequent culture and replating after cryopreservation; n = 10 images, minimum of 2205 individual cells per time point. e After in vitro stratification assay, hPSC-LESCs showed expression of ZO1 tight junction protein and CK3, indicating maturity. f Confocal stacks confirmed expression of p40 at the basal layer of the stratified structure after 3 days of airlifting (shown for hiPSC2-LESCs). Scale bars = 20 μm. BF bright field

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