INTRODUCTION

Primed-type human pluripotent stem cells (PSCs) differentiate into trophoblast (TB) upon treatment with BMP4 plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model). Using this model, we have previously identified nine clusters of TB nuclei by snRNA sequencing, two of which (clusters 5 & 6) were identified as syncytiotrophoblast (STB).

METHODS

Additional downstream analyses of snRNAseq data were performed, including comparative enrichment and gene ontology and pathway analyses. Fluorescent activated nuclei sorting (FANS) was employed with cluster-enriched markers to validate the presence of two kinds of STB nuclei. Immunohistochemistry (IHC) with different combinations of cluster-specific markers was carried out to determine whether these two kinds of STB nuclei co-habit the same STB patch, or whether they represent distinct STB entities.

RESULTS

Genes and pathways related to mitochondria and energy metabolism, translation, and endocrine function were prominent in cluster 5, while cluster 6 was enriched for the AKT/P13K/mTOR metabolic pathways, transcription, and angiogenesis-related pathways. IHC with different combinations of cluster-specific markers uncovered two main classes of syncytial areas as well as some patches of STB with both types of nuclei within a common cytoplasm. By using topoisomerase (TOP1) to distinguish STB nuclei from cytotrophoblast nuclei, and markers enriched in either cluster 5 or cluster 6, nuclei from the two kinds of STB were separated by flow cytometry.

CONCLUSION

These data, combined with previous observations, add to the growing evidence that there may be two kinds of STB.