Introduction: Human amniotic epithelial cells (hAECs), derived from the pluripotent epiblast of the inner cell mass in the blastocyst, form a monolayer of cells covering the placental amnion. While hAECs uniformly exhibit epithelial cell markers, some differentially express stem cell surface markers. We hypothesized that freshly isolated hAECs constitute a heterogeneous population comprising cells at various developmental stages and lineage trajectories. In our previous single-cell RNA sequencing (scRNA-seq) analysis, we identified transcriptional heterogeneity in hAECs; however, we were unable to annotate the cluster identities. Here, we expanded the dataset and performed additional downstream analyses to enhance our understanding of hAEC biology.

Methods: hAECs were isolated from the placentae of a total of five patients who underwent scheduled Caesarean sections. scRNA-seq was conducted using the 10x Genomics Chromium platform. The dataset included three freshly isolated hAEC samples, pooled cultured hAECs collected at multiple time points, and overnight-cultured hAECs. These datasets were integrated for comprehensive analysis. Downstream analyses, including RNA velocity and velocity-pseudotime, were conducted to capture dynamic transcriptional trajectories throughout in vitro culture.

Results: All five datasets were successfully integrated into a single UMAP. Although naïve hAECs formed a compact cluster, five sub-clusters were identified, each characterized by distinct transcriptional profiles suggestive of early ectoderm-, mesoderm-, or endoderm-like lineage signatures. Cultured hAECs exhibited gene expression profiles markedly distinct from those of naïve hAECs, with a transient progenitor-like cluster emerging particularly during the early culture phase. RNA velocity and pseudotime analyses positioned naïve hAECs at the origin of differentiation trajectories, with directional transitions toward more differentiated populations observed throughout culture.

Conclusion: These findings provide updated insights into the heterogeneity and lineage potential of naïve hAECs. These cells appear capable of expressing lineage-associated genes before overt differentiation, supporting their potential to give rise to derivatives of all three germ layers. The expanded dataset enabled us to characterize the rapid transcriptional reprogramming that takes place during the initial phase of hAEC culture. This dynamic transcriptional progression highlighted the plasticity of hAECs and their responsiveness to environmental conditions in vitro.