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

Figure 3.

Figure 3

Characterization of ePE cells

(A) Gene expression of PE markers (HES1, HNF1B, PDX1, FOXA2, ONECUT1, SOX9, and NKX6-1) and endocrine progenitor marker, NEUROG3, in PE and ePE cells derived from HUES4-PDX1-GFP (open-circle dots) and H1 (red dots) ESCs. Direct differentiation indicates PE cells at S4d3 of the Rezania protocol, and ePE cells are categorized by early ePE (P0–P2), late ePE (P5–P10), and polymer ePE (P3–P4) cells. The expression levels were normalized to directly differentiated PE cells at S4d3. The error bars represent the SEM of n = 5 or 3 experimental replicates; ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test comparing the result from direct differentiation PE S4d3 cells with matching ePE cells, as well as from late to early ePE samples.

(B) Representative flow cytometry histograms showing the NKX6-1+ population of PE S4d3 (Rezania protocol), ePE early (P1–P2), and late (P7–P8) passage cells from HUES4-PDX1-GFP (top) and H1 (bottom).

(C) Quantification of NKX6-1+ cells from HUES4-PDX1-GFP (open-circle dots), H1 (red dots), and WTC-11 (gray dots) ePE cells by flow cytometry. The error bars represent the SEM of n = 8 (S4d3 and ePE early) and n = 7 (ePE late) experimental replicates; ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test.

(D) Representative immunostaining images of (top) SOX9 (gray) and PDX1 (red) and (bottom) NKX6-1 (gray) and PDX1 (red), including DAPI (blue), of HUES4-PDX1-GFP ePE cells at P7. Scale bar: 50 μm.

(E) Representative immunostaining images of EdU labeling (gray) and NKX6-1 (red), including DAPI (blue), of HUES4-PDX1-GFP ePE cells at P7. Scale bar: 50 μm.

(F) Representative analysis of the proliferative NKX6-1+ population from ePE cells by flow cytometry. (Left) Representative flow cytometry plot showing NKX6-1 low 25% (green) and high 25% (red) gating of ePE cells among PDX1+ cells. (Right) EdU labeling histograms of NKX6-1 gated populations from the left plot. NKX6-1 high 25% population (red) shows more EdU+ events (23.9%), compared with NKX6-1 low 25% population (6.9%).

(G) Quantification of EdU+ cells among NKX6-1 low and high ePE cells from HUES4-PDX1-GFP (open-circle dots) and H1 (red dots) cells by flow cytometry as shown in (F). The error bars represent the SEM of n = 4 experimental replicates; ∗p < 0.05, Mann-Whitney test.

(H) Karyogram of ePE cells derived from H1 ESCs at P5.