Derivation of new pluripotent stem cells from human extended pluripotent stem cells with formative features and trophectoderm potential

Previous studies have demonstrated the existence of intermediate stem cells, which have been successfully obtained from human naive pluripotent stem cells (PSCs) and peri‐implantation embryos. However, it is not known whether human extended pluripotent stem cells (hEPSCs) can be directly induced into intermediate stem cells. Moreover, the ability of extra‐embryonic lineage differentiation in intermediate stem cells has not been verified. In this issue, we transformed hEPSCs into a kind of novel intermediate pluripotent stem cell resembling embryonic days 8–9 (E8‐E9) epiblasts and proved its feature of formative epiblasts. We engineered hEPSCs from primed hPSCs under N2B27‐LCDM (N2B27 plus Lif, CHIR, DiH and MiH) conditions. Then, we added Activin A, FGF and XAV939 to modulate signalling pathways related to early humans' embryogenesis. We performed RNA‐seq and CUT&Tag analysis to compare with AF9‐hPSCs from different pluripotency stages of hPSCs. Trophectoderm (TE), primordial germ cells‐like cells (PGCLC) and endoderm, mesoderm, and neural ectoderm induction were conducted by specific small molecules and proteins. AF9‐hPSCs transcription resembled that of E8‐E9 peri‐implantation epiblasts. Signalling pathway responsiveness and histone methylation further revealed their formative pluripotency. Additionally, AF9‐hPSCs responded directly to primordial germ cells (PGCs) specification and three germ layer differentiation signals in vitro. Moreover, AF9‐hPSCs could differentiate into the TE lineage. Therefore, AF9‐hPSCs represented an E8‐E9 formative pluripotency state between naïve and primed pluripotency, opening new avenues for studying human pluripotency development during embryogenesis.