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. 2021 Aug 26;12:5140. doi: 10.1038/s41467-021-25237-8

Fig. 1. Formation and characterization of EPI and TS aggregates.

Fig. 1

a Schematic representation of the workflow showing aggregation of ESCs and TSCs on PEG microwells. b Representative images showing EPI aggregates on microwells at 72 h formed from 25 cells/well (top) or 100 cells/well (bottom) showing F-actin staining by phalloidin. Scale bars: 200 µm. c Comparing minimum ferret diameters of EPI aggregates at 72 h formed from 25 or 100 cells/well. For 25 ESC and 100ESC conditions, total number of embryoids analyzed were 362 and 361, respectively. Data are collected from three biologically independent experiments. Large symbols indicate mean values of each replicate. Black lines indicate median and quartiles. d–f Confocal images of EPI aggregates formed from 25 cells/well or 100 cells/well fixed at 72 h and stained for aPKC (d), E-cadherin (e), and F-actin by phalloidin (f). Nuclei were stained with DAPI. Note multilayered epithelium in EPI aggregates formed from 25 cells/well showing discontinuous aPKC staining (white arrows) compared to single-layer epithelium in EPI aggregates formed from 100 cells/well showing continuous aPKC signal. Scale bars: 100 µm. g Confocal images of aggregates formed from 100 cells/well with or without Matrigel fixed at 72 h and stained for E-cadherin and Podocalxyin. Nuclei were stained with DAPI. Scale bars: 100 µm. h, i Confocal images of EPI aggregates formed from 100 cells/well without (h) or with (i) Matrigel, fixed at 72 h showing expression of Otx2, Oct4, Sox2, Nanog, and E-cadherin. Nuclei were stained with DAPI. Scale bars: 100 µm. j Bulk RNA-sequencing analysis of epithelialized and non-epithelialized EPI aggregates at 72 h formed from 100 cells/well, showing expression levels of pluripotency, epiblast-specific and early differentiation genes. Data are collected from four biologically independent experiments. k Representative images showing TS aggregates on microwells at 72 h formed from 25 cells/well (top) or 100 cells/well (bottom) showing ubiquitous GFP expression. Scale bars: 200 µm. l Comparing minimum ferret diameters of TS aggregates at 72 h formed from 25 or 100 cells/well. For 25 TSC and 100TSC conditions, total number of embryoids analyzed were 455 and 478, respectively. Data are collected from four biologically independent experiments. Large symbols indicate mean values of each replicate. Black lines indicate median and quartiles. m Bulk RNA-sequencing analysis of TS aggregates at 72 h formed from 100 cells/well, showing expression levels of stem cell and differentiation markers as well as key pathway agonists/antagonists. Data are collected from four biologically independent experiments. n–p Confocal images of TS aggregates formed from 100 cells/well, fixed at 72 h showing expression of E-cadherin and aPKC (n), Cdx2, Fibronectin, Laminin, and Eomes (o), Cdx2 and Ap2γ (p). Nuclei were stained with DAPI. Scale bars: 100 µm. For statistical analysis, two-tailed unpaired Student’s t-test (c, l) or multiple t-tests followed by Holm-Sidak multiple comparison test (j, m) were performed. For j, False Discovery Rate (FDR) with cutoff p value <0.5 was used to test significant differences between +matrigel and -matrigel conditions. Following P value style was used: (****) < 0.0001, (***) 0.0002, (**) 0.0021, (*) 0.0332, (ns) 0.1234. Source data are provided as a Source Data file.