Journal: Frontiers in Cell and Developmental Biology
Article Title: Integration of human stem cell-derived in vitro systems and mouse preclinical models identifies complex pathophysiologic mechanisms in retinal dystrophy
doi: 10.3389/fcell.2023.1252547
Figure Lengend Snippet: Dram2 loss exacerbates toxicity-induced human RPE cell death in vitro (A) Experimental design of loss of DRAM2 experiments in human cells. hRPE, human primary retinal pigment epithelial (RPE) cells; KD, knockdown; A2E, N-retinylidene-N-retinylethanolamine; NaOI 3 , Sodium Iodate; hPSC, human pluripotent stem cells; KO, Knockout. (B) Representative image of hRPE after infection with lentivirus expressing DRAM2 shRNA and GFP (left) and knockdown efficiency of the two different DRAM2 shRNAs assessed by quantitative RT-PCR (qPCR) analysis of DRAM2 expression (right) (C) hRPE cell survival following treatment with A2E (30 μM; left) and NaIO 3 (5 mM; right). Dotted horizontal line marks the median survival (50% of the cells alive). (D) Differentiation of DRAM2 WT and KO human pluripotent stem cells (hPSCs) into RPE (hPSC-RPE) showing pigmentation (top row, BF: Bright Field) and expression of RPE maker ZO-1 (bottom row). Scale bars: 50 µm. (E) Representative immunofluorescent image (left) of RPE marker ZO-1 (green) in hPSC-RPE DRAM2 WT, KO1, and KO2 and phagocytosis of FITC-labeled Photoreceptor Outer Segments (POS; red). Scale bar: 20 µm. Phagocytosis capacity of the cells was assessed by quantification of cells with internalized FITC-POS by flow cytometry ( DRAM2 WT: blue line, DRAM2 KO1 and 2: orange lines). (F) hPSC-RPE cell survival following treatment with A2E (30 μM; left) and NaIO 3 (5 mM; right). Dotted horizontal line marks the median survival.
Article Snippet: Human primary retinal pigment epithelial (hRPE) cells (Lonza #00194987) were maintained in Retinal Pigment Epithelial Cell Growth Medium (RtEGM; Lonza) per manufacturer protocols.
Techniques: In Vitro, Knock-Out, Infection, Expressing, shRNA, Quantitative RT-PCR, Marker, Labeling, Flow Cytometry