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. 2020 Feb 25;8:109. doi: 10.3389/fcell.2020.00109

FIGURE 1.

FIGURE 1

Assessment of the pancreatic islet hormones expression by global proteomics and large-scale imaging following three distinct differentiation strategies (A) Experimental design depicting the three differentiation strategies considered. Numbers in bold represent differentially expressed proteins (DEPs) between the final differentiation stage (S7, S7enc[S5–S7], 2w_postTX) and pancreatic progenitors (S5), following islet-standard normalization (data represent two distinct TMT-11 plexes). (B) Graphs displaying the statistically significant (FC ≥ 1.5, p < 0.05) regulated islet hormones (colored bars) identified in the DEPs set characterizing each differentiation strategy (in vitro Matrigel differentiation – chartreuse, in vitro encapsulation – yellow and in vivo transplantation of encapsulated cells – light green). (C) 3D reconstructions of dragonfly imaged whole alginate capsules containing cells immunofluorescently labeled for insulin (green), glucagon (red) and DAPI (blue) in the four conditions analyzed (scale bar 200 μm, gamma correction 0.4). (D) Proportion of monohormonal glucagon cells in the four distinct populations analyzed (n = 9,18,18,18) and the high magnification of a representative encapsulated glucagon+ cell. (E) Proportion of monohormonal insulin in the four distinct populations analyzed (n = 9,18,18,18) and the high magnification of a representative encapsulated insulin+ cell. (F) Proportion of bihormonal cells (insulin+glucagon+ co-expressing) and a high magnification of a representative encapsulated bihormonal cell. High magnification scale bars: 5 μm. Graphs data are shown as box plot mean to max values. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Mann–Whitney test). Abbreviations: TX, transplant; 2D, Matrigel differentiation; 3D, alginate encapsulation; DEPs, differentially expressed proteins; Dt, time interval; FC, fold change; S5-cells, stage 5 cells (pancreatic progenitor stage); S7-cells, stage 7 cells (maturing β-cells); S7enc[S5–S7], differentiated in capsules from stage 5 to stage 7; 2w_postTX, 2 weeks following transplantation (differentiation in mice).