Fig 2: Cell line–dependent gene expression regulates pluripotency and initial lineage commitment.(A) Heatmaps with expression patterns of gene modules associated with regulators of pluripotency: SOX2, POU5F1, PRDM14, KLF4, and NANOG. The modules are determined with dynamic tree cutting of hierarchical clusters with respect to gene expression across all time points and cell lines. Each column of the heatmap represents a cell line, from left to right: T, TL; S, SCVI15; L, LEPCC3 (asterisk marks promyogenic line L*). Within each of the three columns, eight boxes represent time point days 3 to 30. Heatmap colors: Blue represents down-regulated and red represents up-regulated L2FC gene expression with respect to each cell line’s respective day 0 undifferentiated value. (B) Heatmaps with expression patterns of gene modules associated with key regulators of differentiation: T and EOMES are crucial regulators of mesendoderm specification, while HOXA2, HOXB5, and HOXD10 are representative of anterior to posterior HOX family genes. (C) Heatmap detailing the noncoding RNA (ncRNA) transcripts that cluster near NANOG: red, known lncRNAs; green, unannotated transcripts. (D) Heatmap detailing the noncoding ncRNA transcripts that cluster near HOXD9: red, known lncRNAs; green, unannotated transcripts.

Fig 3: Human pluripotent stem cells efficiently form endoderm and hepatic progenitor cells in vitro. (A) RT-qPCR analysis of RNA extracted from UD, EP, HP, and ectoderm (ECT) cells displayed as delta-delta Ct values relative to HMBS “housekeeping gene product” (n = 3 biological replicates shown as individual points, bars show mean, and error bars show standard deviation). (B) Representative images of indirect immunofluorescence of UD H9 ESCs showing SOX2 (magenta), SSEA3 (yellow), DAPI (blue), and each channel overlaid; of EP cells showing SOX17 (magenta), DAPI (blue), and the two channels overlaid; and HP cells with AFP (magenta), DAPI (blue), and each channel overlaid. Scale bar denotes 100 µm. (C) Flow cytometry analysis of EP cells (top) stained with either isotype control antibody: APC conjugate (gray histogram) or anti-CXCR4: APC conjugate (magenta histogram), and HP cells (bottom) stained with either isotype control antibody: APC conjugate (gray histogram) or anti-CD99: APC conjugate (cyan histogram). Histograms show a result representative from four biological replicates; mean values for percent positive are shown ±standard deviation. (D) Western blot analysis of total protein extracted from UD, EP, CM, ECT, or HP cells and probed with antibodies directed against EOMES, SOX17, NKX2.5, AFP, and Tubulin; results shown are representative of at least two biological replicates. CM: cardiac mesoderm; DAPI: 4’,6-diamidino-2-phenylindole; EP: endoderm progenitor; HP: hepatic progenitor; UD: undifferentiated hESCs.

Fig 4: Histological characterization of uncut and cut human pluripotent stem cell-derived cerebral organoids (hCOs) Histologic characterization of (A) uncut and (B) cut hCOs conducted using immunohistochemical staining. SOX2, sex-determining region Y-box 2 (neural progenitor marker, red), TUJ1, neuron-specific class III beta-tubulin 1 (neuronal marker, green), PAX6, paired box 6 (radial glial cell marker, red), TBR2, T-box brain protein 2 (intermediate progenitor cell marker, green), neuronal-specific N-cadherin (green), DCX, double cortin (neuronal marker, green). Nuclei were stained with DAPI (4',6-diamidino-2'-phenylindole). Scale bar:100 µm.