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. 2021 Aug 11;70(11):2554–2567. doi: 10.2337/db20-0873

Figure 1.

Figure 1

sBC self-enrich to form insulin+ enriched islet-like caps. A: Schematic representation of stepwise differentiation of human pluripotent stem cell clusters toward β-like cells in suspension. B: Representative live image of green fluorescent protein driven by the endogenous insulin promoter (pINSGFP) of clusters during imBC (day ∼23) and seBC (day ∼30) differentiation stages (scale bars indicate 200 μm). C: Flow cytometric quantification of GFP expression in imBC and seBC clusters (n = 7 independent differentiation experiments). D: Quantification of GFP intensity in imBC and seBC clusters (n = 6 independent differentiation experiments). E and F: Immunofluorescence analysis of sections from imBC and seBC clusters, respectively, for endocrine and β-cell markers. G: Immunofluorescence analysis of sections from imBC clusters, seBC clusters, and human islets for mitochondria-specific mtFA protein. H: Quantification of mtFA fluorescence intensity in imBC and seBC cells (n = 5 independent differentiation experiments with 10 clusters analyzed per experiment). I: Schematic representation of pINSGFP+ cell sorting from imBC and seBC clusters. J: Bulk RNA-seq analysis of pINSGFP+ sorted imBC vs. seBC. Top 30 genes significantly up- and downregulated with adjusted P value <0.05 are shown (n = 3 independent differentiation experiments). K: GO of differentially regulated genes. L: Quantitative PCR analysis of mtDNA normalized to gDNA in pINSGFP+ sorted cells (n = 3 independent differentiation experiments with 3 × 500 cells collected for analysis from each). M: Global levels of 5-hmc in pINSGFP+ sorted cells (n = 3 independent differentiation experiments). N and O: Total insulin content (N) and proinsulin-to-insulin content ratios (O) per 1,000 pINSGFP+ sorted cells (n = 3 independent differentiation experiments with 3 × 1,000 cells collected per experiment). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars are representative of the mean ± SD. Scale bars represent 20 μm unless otherwise indicated. AU, arbitrary units; ns, not significant.