Transcriptome comparisons of regenerative trophoblast models are largely in concordance with trophoblasts from peri-implantation human embryos while maintaining subtle differences.

Understanding the differentiation of early trophoblasts (TB) during the peri-implantation period is crucial to understanding pathologies related to TB invasion, including pre-eclampsia and recurrent implantation failure. Human TB cell lines are the most accessible models to study early placental development, yet their ability to faithfully mimic the identity of peri-implantation TB has not been directly examined. We aimed to evaluate to what extent modern regenerative TB models are able to represent TB in the primitive placenta during implantation. We used RNA sequencing to define the transcriptomic landscape of three TB cell lineages, cyto-TB (CTB), syncytio-TB (STB), and extravillous-like migratory TB (MTB) from two widely used cell models, BMP4/A 83-01/ PD173074-treated embryonic stem cells (H1 and H9, BAP) and true human TB stem cells (CT-27, CT-29, TSC), and directly compare them with TB from human embryos cultured during the peri-implantation period up to embryonic day 8, 10, and 12 in vitro. RNA-seq libraries were constructed following SMART-Seq v4 protocol (Takara Bio) and sequenced on Illumina NovaSeq 6000 platform. All conventional analyses, including Principal Component Analysis (PCA) and clustering analysis, were performed based on the R platform. The gene ontology and pathway analysis were performed using the David tool. We also identify novel TB marker transcripts upregulated at least 4-fold across each trophoblast model and use immunofluorescence to validate their presence in peri-implantation human embryos. All human embryo work was completed using embryos donated to research following IVF treatment and all experiments were approved by the WCGIRB. TB sublineages from different models grouped together following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences amongst models exist for each TB sublineage. TSC and early-stage day 8 EEC CTB upregulate transcripts related to increased stem cell renewal compared to BAP and later-stage day 10 and 12 EEC CTB. In STB, EEC TB are enriched in GO terms related to oxygen-dependent metabolism, while highly enriched pathways from BAP and TSC are related to lipid metabolism, matrix remodeling, and protein processing. Finally, we provide a pool of potential lineage markers in early TB for each subpopulation that were upregulated across all three models and validate the presence of ANLN, FIBCD1, and MAMDC2 in peri-implantation stage human embryos. Our comprehensive analysis revealed that both commonly used BAP and TSC models resemble features of peri-implantation TB, while still maintaining subtle transcriptomic differences yet to be explored functionally.