Journal: Scientific Reports

Article Title: Human cell dedifferentiation in mesenchymal condensates through controlled autophagy

doi: 10.1038/srep13113

Figure Lengend Snippet: ( a ) Analysis of 3D MSC formation and size over 7 days in culture. Left panel, diameter mean values shown ± SD, n = 6; right panel micrographs of whole spheroids, (scale bar = 250 μm). 30, 000, 60, 000 and 120, 000 refer to initiating cell number. ( b ) Expression of Oct4, Nanog and Sox2 determined by quantitative polymerase chain reaction (QPCR) in different sized spheroids over time in 3D culture. Data represent three separate experimental donors (n = 3) and are shown as mean values ± SEM, * = p < 0.05. 30, 000, 60, 000 and 120, 000 refer to initiating cell number. ( c ) Immunofluorescent detection of EdU incorporation in 3D MSCs to identify proliferating cells (2D MSCs are shown as a positive control), scale bar = 50 μm. ( d ) QPCR analysis of pluripotency factors following disaggregation of 3D MSCs and return to 2D culture. Data represent three separate experimental donors (n = 3) and are shown as mean values ± SEM, * = p < 0.05. ( e ) Effects of 3D culture on morphology and size of MSCs that had been in vitro -aged to induce senescence-associated hypertrophy through extended time in culture. Brightfield image (left), darkfield image (middle) with arbitrary colour mask using CellProfiler software (right) to aid visualiation (scale bar = 100 μm). ( f ) QPCR analysis of Oct4, Nanog and Sox2 in 3D MSCs and 2D MSCs compared to ESCs. Data represent three separate experimental donors (n = 3) and are shown as mean values ± SEM, * = p < 0.05, **** = p < 0.0001. See also .

Article Snippet: Mouse ESCs (E14) included as positive controls were cultured and prepared by Reinnervate Ltd. All work was carried out in accordance with Home Office ethical guidelines.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Positive Control, In Vitro, Software

Journal: Scientific Reports

Article Title: Human cell dedifferentiation in mesenchymal condensates through controlled autophagy

doi: 10.1038/srep13113

Figure Lengend Snippet: ( a ) QPCR analysis of markers of early mesendoderm in 3D MSCs (expression normalized to GAPDH, fold changes calculated as 2(-DDCt)) compared to 2D MSCs. Data represent two separate experimental donors (n = 2) and are shown as mean values ± SEM, * = p < 0.05, *** = p< 0.001. ( b ) Immunofluorescent detection (red fluorescence) of Brachyury and CXCR4 expression in 2D MSCs (top panel) 3D MSC condensates (bottom panel) (scale bar = 100 μm). Nuclei identified by DAPI staining (blue fluorescence). ( c ) In vivo tissue formation assay using 3D MSC condensates, tissues labelled as muscle (Mu), cartilage (Ca), adipose (Ad) and connective tissue (Co). Sections stained with hematoxylin and eosin (H and E) and Masson’s Trichrome. All micrographs show tissues formed by 3D MSC condensates. Scale bars = 500 μm (far left panel only) and 100 μm. ( d ) In vivo tissue formation assay using mouse ESCs, germ layers identified as Mesoderm (Mes), Endoderm (End) and Ectoderm (Ect), sections stained with hematoxylin and eosin ( H,E ) and Masson’s Trichrome. All micrographs show teratomas formed by mouse ESCs. Scale bars = 500 μm (far left panel only) and 100 μm. See also .

Article Snippet: Mouse ESCs (E14) included as positive controls were cultured and prepared by Reinnervate Ltd. All work was carried out in accordance with Home Office ethical guidelines.

Techniques: Expressing, Fluorescence, Staining, In Vivo, Tube Formation Assay