Introduction: Human amniotic epithelial cells (hAECs) are a type of placental stem cell that possesses the ability to differentiate into all three germ layers. It has been reported that primary hAECs homogeneously express epithelial cell markers while they heterogeneously express several pluripotent stem cell surface markers. Furthermore, we previously showed that primary hAECs underwent epithelial-mesenchymal transition and differentiated immediately after isolation from the placenta. These findings suggest that some of the epiblast-derived hAECs initiate spontaneous differentiation into multiple lineages during the gestation period, and thus primary hAECs contain a population of cells in varying degrees of differentiation. In this study, we aimed to investigate the phylogenetic diversification of hAECs using single-cell RNA sequencing (scRNA-seq).

Methods: hAECs were isolated from the placentae of four patients who underwent scheduled Caesarean sections. scRNA-seq was performed on freshly isolated hAECs (n=3, unpooled) and cultured hAECs (pooled from several culture durations) using a 10x Genomics Chromium system. To determine lineage-specific traits, we referenced the expressions of 27 genes for endoderm, 27 genes for mesoderm, and 26 genes for ectoderm according to a lineage marker genes panel based on ES and induced pluripotent stem cell studies.

Results: Similar patterns of gene expression were observed among freshly isolated hAECs despite being derived from different donors. Cluster analysis by Uniform Manifold Approximation and Projection (UMAP) plot showed that the primary hAEC population was not clearly separated into multiple independent clusters. However, distinguished patterns of gene expression were detected even within this seemingly homogeneous population. A group of hAECs that differentially expressed pluripotency marker genes such as POU5F1 were also identified. Furthermore, single-cell analysis of cultured hAECs allowed us to identify representative genes for each germ layer based on their expression levels, revealing the spontaneous differentiation with phylogenetic diversification of hAECs.

Conclusion: This is the first study that unraveled the hAEC heterogeneity at the single-cell transcription level resolution. We identified subgroups of hAECs with diverse differentiation tendencies, allowing for the selection of subgroups that are suitable for different clinical applications. Moreover, in silico reconstruction of the gene expression patterns has the potential to contribute to our fundamental understanding of complex cell differentiation during human placentation.