Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability

Figure 3

Global proteomics of the 20 reprogrammed cell lines reveals different proteomic signatures for the different colony morphology groups. (a) Workflow for the proteomic experiment analyzing the global proteome of 20 reprogrammed cell lines microscopically classified into stable colony morphology (14 lines), unstable class 1 colony morphology (4 lines), and unstable class 2 colony morphology (2 lines). The samples are cell lysates from the corresponding cell line. The samples were quantified using label-free proteomics, which yielded ~5000 proteins in each sample. Raw values were log₂ transformed, and fold changes (FC) between the combined unstable colony morphology group (6 lines) and stable colony morphology group (14 lines) were calculated. Proteins with a value < 0.05 and a FC value < 0.05 () were regarded as being differentially abundant between the groups. (b) Unsupervised clustering analyses of the 20 reprogrammed lines based on proteins expressed in at least 14/20 samples () showing that the clusters tend to associate more with morphology and less with donor sex and reprogramming methods. S = stable colony morphology; U = unstable colony morphology. (c) Principal component analysis (PCA) of proteins expressed in all samples () shows a clear separation between stable colony morphology (black circles) and unstable colony morphology (grey and white circles). (d) Unsupervised clustering analyses of the 614 differentially abundant proteins. The clustering analysis revealed two major clusters separated by colony morphology. (e) Tables describing molecular and cellular functions of the protein being more abundant in the unstable colony morphology group () and the stable colony morphology group (). (f) A selection of differentially abundant proteins in the morphology groups. EMT markers (VIM, MMP14, FN1, and CDH2) and ectoderm markers (NES, MAP2) were more abundant in the unstable colony morphology group (U), whereas pluripotency markers (PODXL, DPPA4, DNMT3B, and CDH1) were more abundant in the stable colony morphology group (S). Statistical analysis was performed with Mann-Whitney -test. Significant differences are shown as , , , , and ns . n/a = not a number. (g) Top differentially abundant proteins ranked by fold change (stable compared to unstable). Only proteins with a value < 0.05 were included in the ranking. The pluripotency markers DNMT3B, DPPA4, and SALL4, the EMT marker FN1, and the ectoderm marker MAP2 are highlighted in green (up in the stable colony morphology signature) and red (up in the unstable colony morphology signature).

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