Fig. 3: Expansion and enrichment of monolayer differentiated hPSC-RPE on hrLN-521. | Nature Communications

Fig. 3: Expansion and enrichment of monolayer differentiated hPSC-RPE on hrLN-521.

From: Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

Fig. 3

a hPSC-RPE monoloayer differentiation protocol scheme (top image was adapted from ref. 10, Copyright (2016), with permission from Elsevier). hESC cultures are seeded on hrLN-521- or hrLN-111-coated plates in NutriStem hPSC XF medium containing Rho-kinase inhibitor (Y-27632) in 5% O2. After 24 h, the medium is replaced with differentiation medium (NutriStem hPSC XF without bFGF and TGFβ) and cells are placed in 21% O2. From day 6 after plating, 100 ng/mL of Activin A (R&D Systems) is added to the media until day 30. Cells are then enzymatically dissociated, replated on hrLN-521, and cultured until homogeneous pigmentation is reached (day 60). At that point, single-cell suspension is injected subretinally in the rabbit eye. b Bright field and immunofluorescent images showing the co-expression of RPE-specific markers (BEST-1, MITF, CRALBP) with CD140b in hPSC-RPE day 60. c Flow cytometry for TRA-1-60 and CD140b following replating at cell densities: 1.4 × 106 cells/cm2 (1:1), 7 × 104 cells/cm2 (1:20), 2.8 × 104 cells/cm2 (1:50), and 1.4 × 104 cells/cm2 (1:100). d Relative cell yield during the differentiation protocol at various replating densities. e Gene expression analysis of pluripotency, RPE- and neural-specific genes normalized to GAPDH and relative to hESC. f Functional assays demonstrating epithelial integrity by transepithelial resistance (TEER), PEDF secretion by ELISA, and internalization of photoreceptor outer segments (POS). The TEER value for hESC is shown for comparison (dashed line). g SEM and TEM images of hESC-RPE cultured on hrLN-521 at two different magnifications showing surface microvilli and polarized intracellular structures. h Pigmented monolayer formation in albino rabbits demonstrated by bright field imaging and immunofluorescence of anti-human BEST-1 and NuMA upon subretinal injection of hPSC-RPE cultured in hrLN-521 (replated at a cell density of 7 × 104 cells/cm2). Bars represent means ± SEM from three independent experiments. (*) Asterisks represent significance with a P value < 0.0001 (3F, TEER, and PEDF secretion); =0.0042 (3F, Phagocytosis). Scale bars: b = 20 μm; g = 10 μm; h = 50 μm. Source data are provided as a Source Data file.

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