Abstract

Objectives This study aims to optimize the human extended pluripotent stem cell (EPSC) to trophectoderm (TE)-like cell induction with addition of EGF and improve the quality of the reconstructing blastoids. Materials and Methods TE-like cells were differentiated from human EPSCs. RNA-seq data analysis was performed to compare with TE-like cells from multiple human pluripotent stem cells (hPSCs) and embryos. A small-scale compound selection was performed for optimizing the TE-like cell induction and the efficiency was characterized using TE-lineage markers expression by immunofluorescence stanning. Blastoids were generated by using the optimized TE-like cells and the undifferentiated human EPSCs through three-dimensional culture system. Single-cell RNA sequencing was performed to investigate the lineage segregation of the optimized blastoids to human blastocysts. Results TE-like cells derived from human EPSCs exhibited similar transcriptome with TE cells from embryos. Additionally, TE-like cells from multiple naive hPSCs exhibited heterogeneous gene expression patterns and signalling pathways because of the incomplete silencing of naive-specific genes and loss of imprinting. Furthermore, with the addition of EGF, TE-like cells derived from human EPSCs enhanced the TE lineage-related signalling pathways and exhibited more similar transcriptome to human embryos. Through resembling with undifferentiated human EPSCs, we elevated the quality and efficiency of reconstructing blastoids and separated more lineage cells with precise temporal and spatial expression, especially the PE lineage. Conclusion Addition of EGF enhanced TE lineage differentiation and human blastoids reconstruction. The optimized blastoids could be used as a blastocyst model for simulating early embryonic development.