Fig. 4. Progression from naïve- to primed-pluripotency in EPI-like compartment.

a The Epi-like compartment of RtL-embryoids shows three transcriptionally diverging subpopulations, assessed in UMAP representation. b Comparison to Anterior-, Transition-, and Posterior-Epiblast signatures published by Cheng et al.²³ revealed highest transcriptional similarity with anterior-epiblast cells. c Marker gene expression for Anterior-, Transition-, and Posterior-marker genes. d, f IF staining against pluripotency markers during progression from rosette- to lumen stage. The Epi-like compartment of RtL-embryoids (indicated by dotted lines) displays downregulation of naïve-pluripotency markers KLF4 and ESRRB during progression from rosette to lumen stage. Expression of OTX2 was detected at both, rosette and lumen stage. Scale bars = 50 µm. KLF4/ESRRB/OTX2, red; Phalloidin (Phall)/ PODXL, yellow; DAPI, blue. g Primed-pluripotency marker OCT6 was detected to be weakly expressed in some OTX2 + cells at lumen stage; Lower Panel shows magnification of area indicated in panel above. Scale bars = 50 µm. OTX2, red; OCT6, green; PODXL, yellow; DAPI, blue. h Expression of OCT4 and NANOG was detected in RtL-embryoids throughout the culture period. Scale bars = 50 µm. OCT4, green; NANOG, red; DAPI, blue. i pERK pulses were detected in single Epi-like cells of RtL-embryoids at lumen stage, in addition to a diffuse and weak pERK activity in the ExE-like compartment. Scale bars = 50 µm. OTX2, red; pERK, green; DAPI, blue. White arrow indicates pERK+ Epi-like cell. j Single-cell heatmap of core-, naïve-, and primed- pluripotency markers among cells of the Epi-like Subclusters, revealing predominantly naïve-pluripotency factor expression in subcluster 1, while subcluster 2 displayed downregulation of naïve- and upregulation of primed-pluripotency factor in subcluster 2. Subcluster 3 displayed a PGC-like character. Experiments were repeated independently at least three times with similar results (d–i).