Fig. 5: Multi-lineage reprogramming and differentiation confirms that TNT reprogramming enhances differentiation.
From: Transient naive reprogramming corrects hiPS cells functionally and epigenetically
a, Experimental design for multi-lineage primed and TNT reprogramming and differentiation into five cell types. Top, the four somatic cell lines reprogrammed into primed-hiPS cells and TNT-hiPS cells with three independent reprogrammings (r1–r3) performed per group, and with each subsequently differentiated into five different cell types, with independent replication. Bottom, the number of independent differentiation replicates performed for origin cell types (rows) and differentiated cell types (columns). Coloured circles represent primed-hiPS cell (green), TNT-hiPS cell (yellow) and hES cell (grey). 2° fibroblasts, secondary fibroblasts. b, Endoderm differentiation quantification for hiPS cells derived from secondary fibroblasts, showing the proportion of cells positive for FOXA2 and SOX17 by immunofluorescence analysis. c, Representative images from immunofluorescence analysis of FOXA2 and SOX17 in endoderm differentiation of hiPS cells derived from secondary fibroblasts. The outlined region is enlarged on the right. Scale bars, 100 μm (main image), 50 μm (enlarged region). d, Quantification of multi-lineage cell differentiation in hiPS cell lines by FACS and immunofluorescence analyses using CD56, CD57 (FACS), PAX6 and SOX1 (immunofluorescence) for cortical neuron differentiation, CD146, CD56 (FACS), PAX3 and PAX7 (immunofluorescence) for skeletal muscle differentiation, and CD47, EPCAM (FACS), GATA6 and TTF1 (immunofluorescence) for lung epithelial differentiation. e, Representative images from immunofluorescence analysis of cell differentiation using SOX1 and PAX6 for cortical neurons, PAX3 and PAX7 for skeletal muscle, and GATA6 and TTF1 for lung epithelial cells. Scale bars, 50 μm. f, Phase-contrast images taken four days after passaging plated embryoid bodies during differentiation into NSCs. Large stretched-out fibroblast-like cells are evident during differentiation from primed-hiPS cells (red arrows). g, The percentage of NCAM+FAP− cells (from FACS analysis) after plating of embryoid bodies during NSC differentiation. log2FC values are shown on the graph. d,g, Data are mean ± s.d; two-sided t-test for primed versus TNT; ***P < 0.0001, **P < 0.001, *P < 0.05. Details of replication are presented in Methods, ‘Statistics and reproducibility’.
