Fig. 1
From: AAV vector-mediated in vivo reprogramming into pluripotency
In vitro reprogramming of MEF using AAV-DJ SFFV-hCO-O/K/S/M vectors. a Experimental setup. MEF were transduced at days 1 and 3 after plating with the four vectors, using a 4:1:1:1 O:K:S:M stoichiometry and one of the two indicated MOIs per experiment and per vector (with a four-fold excess of Oct-3/4). At day 5, cells were transferred from a well of a 6-well plate to a 10 cm dish. Subsequently, medium was changed every other day to iPSC medium with or without ascorbic acid (AA). First colonies with iPSC morphology were observed at day 13. The image shows a representative alkaline phosphatase staining of clone IVT-iPSC 12 at passage 1. n = 4 for MOI 1 × 104 and n = 3 for MOI 1 × 103. b Confocal images exemplifying expression of Oct-3/4, Sox2, Nanog and SSEA-1 in AAV-derived iPSC (again IVT-iPSC 12). Shown are merges of Hoechst and secondary antibody (Alexa Fluor 488-labeled) stainings. Scale bars = 20 µm. c Reverse transcription-PCR analysis to detect expression of the depicted pluripotency markers. Shown are 10 representative clones from the reprogramming series with ascorbic acid. See Supplementary Data 1 (tab “In vitro”) for details. Six of the ten shown clones (IVT-iPSC 19 to 22, 24, and 26) are positive for all markers. Hprt (Hypoxanthine-guanine phosphoribosyltransferase) served as housekeeper. C+ iPSC generated with a lentiviral vector encoding human codon-optimized OKSM (positive control); C− untreated MEF (negative control); P, passage number. d Confocal images showing expression of ectodermal, mesodermal, and endodermal markers in differentiated clone IVT-iPSC 1. Shown are merges of Hoechst and secondary antibody (Alexa Fluor 488-labeled) stainings. Scale bars = 13 µm. Confocal images in b and d were taken with a Leica SP5 microscope with a ×40 oil immersion objective and processed with ImageJ. e Hematoxylin/eosin staining of a section of a subcutaneous teratoma generated in mice via injection of clone IVT-iPSC 1. Structures belonging to the three embryonic germ layers are indicated by black arrows. Scale bar = 100 µm. This figure contains elements from Servier Medical Art (http://smart.servier.com)
