Fig. 2: Choanoflagellate Sox can induce pluripotency in mammalian cells.
From: The emergence of Sox and POU transcription factors predates the origins of animal stem cells
a Schematic illustration of the procedure of mouse induced pluripotent stem cell (iPSC) reprogramming from mouse embryonic fibroblasts (MEFs) carrying an Oct4-GFP reporter (OG2MEFs) and the establishment of clonal iPSC line for pluripotency validation. b Representative microscope images show iPSC colonies generated by mSox2 and Sox factors of Choanoflagellates on reprogramming day 14. Scale bar, 80μm. Chimeric-Salhel-I (Chimeric-Salhel-Sox-I), HMG of Salhel-Sox-I fused with mSox2 NTD and CTD; Salhel-Sox-I, full-length Salhel Sox-I; Salhel-Sox-II, full-length Salhel Sox-II; Myflu-Sox-I, full-length Myflu Sox-I; Myflu-Sox-II, full-length Myflu Sox-II. c Quantification of iPSC reprogramming efficiency by Sox variants. The heatmap depicts the number of experiments with observation of GFP-positive colonies, with the red frame highlighting the ability of the variants to produce iPSCs with confirmed pluripotency through the establishment of stable clonal iPSC lines. Chim-Salhel-I, Chimeric-Salhel-Sox-I; Chim-Pchi, Chimeric-Pchi-Sox. The box plot shows the reprogramming efficiency of Sox variants normalized by the number of iPSC colonies generated by mSox2. (n = 7 technical replicates in total, 2 biological replicates each with 2 technical replicates and 1 biological replicates including 3 technical replicates). The box displays the interquartile range, with the left edge representing the lower quartile (25th percentile) and the right edge indicating the upper quartile (75th percentile). The median value is shown as a line splitting the box. The silhouettes of the species are sourced from PhyloPic (http://phylopic.org). d Representative images of iPSC colonies derived from MEFs carrying a Sox2-GFP reporter on reprogramming day 14. Scale bar, 80 μm. e Expression of pluripotency markers of clonal iPSC lines derived by choanoflagellate Sox examined by immunocytochemistry staining. Scale bar, 40μm. f Immunocytochemistry of differentiated iPSC lines stained for markers of the 3 germ layers: Class III beta-tubulin (Tuj1), Forkhead box protein A2 (FoxA2), α-smooth muscle actin (SMA). Scale bar, 40 μm. g Chimeric mice generated from full-length Salhel-Sox-I iPSC lines displaying black coat patches and eyes (indicated by arrows) representing their iPSC origin, in contrast to the wildtype mouse exhibiting a white coat and red eyes. The illustration for (a) was created in BioRender. Gao, Y. (2022) BioRender.com/z21g065. d n = 2 replicates; (e, f) n = 3 replicates. Source data and statistics are provided as a Source Data file.
