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. 2022 Apr 21;17(5):1215–1228. doi: 10.1016/j.stemcr.2022.03.013

Figure 5.

Figure 5

Endocrine differentiation of ePE cells

(A) Schematic diagram of endocrine cell differentiation from PE S4d3 and ePE cells at P5, P7, or P10 in planar culture or on the air-liquid interface.

(B) Gene expression profile of CHGA and AMY from HUES4-PDX1-GFP (open symbols) and H1 (red symbols) cells at S6d15 differentiation, from either directly differentiated or ePE cells (FN at P5–P10 or polymer at P3). Samples were normalized to directly differentiated samples, and ePE cells (n = 5) were used as a negative control. Error bars represent the SEM of n = 9 or 12 experimental replicates; ∗∗∗∗p < 0.0001, Mann-Whitney test comparing the data of ePE differentiation with those of direct differentiation.

(C) Gene expression profile of NKX6-1, INS, MAFA, GCG, and SST from HUES4-PDX1-GFP (open) and H1 (red) cells at S6d15 differentiation, from either directly differentiated or ePE cells (FN at P5–P10 or polymer at P3). Samples were normalized to directly differentiated samples. Human islets (n = 3) and ePE cells (n = 5) were used as positive and negative controls, respectively, for endocrine genes. Error bars represent the SEM of n = 9 or 12 experimental replicates; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Mann-Whitney test comparing the data of ePE differentiation at S6d15 with those of direct differentiation.

(D) Representative immunostaining images of NKX6-1, INS, GCG, C-PEP, and MAFA (as color-coded on the corresponding lines) for ePE (P9) differentiation at S6d15. Scale bar: 50 μm.

(E) Representative flow cytometry plots showing INS and GCG staining in populations of direct differentiation and ePE differentiation at S6d15.

(F) Quantification of INS+ and GCG+ cells in populations of direct differentiation (gray bars) and ePE differentiation (white bars) at S6d15 at the air-liquid interface with legend shown in (B). The error bars represent the SEM of n = 9 experimental replicates; ∗p < 0.05, Mann-Whitney test comparing the data of ePE differentiation at S6d15 with those of direct differentiation.

(G) Functional characterization of insulin-producing cells generated from ePE (open-circle dots) or direct differentiations (black dots). Sequential static glucose-stimulated insulin secretion (GSIS) assay comparing the insulin secretion of ePE or directly differentiated aggregates at S6d15 challenged with low (2.8 mM) and high (20 mM) glucose in the interval of 1 h and also with 3-Isobutyl-1-methylxanthine (IBMX) + forskolin and KCl in the interval of 30 min. The dot plot shows insulin content in 1,000 cells for each condition. Error bars represent the SEM of n = 6 or 4 experimental replicates, two technical replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, paired two-tailed t test comparing the data of insulin secretions with treatment with high glucose (20 mM), high glucose with IBMX + forskolin, or high glucose with KCl with low glucose (2.8 mM) stimulation; unpaired two-tailed t test comparing the data of each treatment between ePE and direct differentiation.