Characterization of epigenetic regulation during early trophoblast differentiation in human expanded potential stem cells

Abstract

Proper early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Emerging evidence suggests that epigenetic activation of differentiation-associated genes and silencing of self-renewal-associated genes occurs at the onset of first lineage segregation. It was hypothesized that early trophoblast differentiation was epigenetically regulated through pre- and post-transcriptional modifications of trophoblast determinants. Taking advantage of a morula-like human expanded potential stem cells derived from preimplantation embryos (hEPSC-em), this study aimed to decipher the roles of epigenetic regulations through DNA methylation and non-coding microRNA (miRNA) in early trophoblast development. To allow fast lineage tracing of trophoblastic differentiation, the early trophoblast marker GATA3 was selected as the reporter to generate hEPSC-em-GATA3mCherry reporter line by CRISPR/Cas9 approach. A homozygous reporter line was successfully established without disrupting the pluripotency and differentiation potential of hEPSC-em. The differential expression patterns of DNA methylation writers (DNA methyltransferases, DNMTs) and erasers (ten-eleven translocation methylcytosine dioxygenases, TETs) were analysed by bioinformatic analysis of published datasets of pre- and post-implantation embryos. While DNMT3A, DNMT3B and TET1 were downregulated, TET2 and TET3 were upregulated during early trophoblast differentiation. Similar patterns of DNMTs and TETs were observed in hEPSC-em derived trophoblastic spheroids (BAP-EB). Moreover, the treatment of DNMT inhibitor improved the efficiency of trophoblast differentiation, whereas TET inhibitor impeded the differentiation process. Further integrative dataset analyses identified aminopeptidase A (ENPEP), a TE progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Follow-up molecular and functional studies demonstrated that ENPEP was activated by TET2-mediated DNA demethylation during trophoblast differentiation. ENPEP knockout in hEPSC-em led to impairment in trophoblast differentiation with reduced adhesion and invasion of BAP-EB, indicating its vital roles in trophoblast fate commitment. This study further delineated the roles of Argonaute2 (AGO2), the core component of miRNA-induced silencing complex, in early trophoblast development. While AGO2 knockout (AGO2-KO) in hEPSC-em cell had no impact on its pluripotency, hEPSC-em devoid of AGO2 failed to establish proper TSC and induced amniotic (AME) signature during TSC-derivation. The AGO2-KO TSC also had impaired differentiation potentials into syncytiotrophoblast (STB) and extravillous trophoblast (EVT). Single-cell RNA sequencing followed by gene ontology analysis also revealed that the upregulated genes in AGO2-KO TSC and STB were enriched for AME-related pathways. A modified Covalent Ligation of Endogenous Argonaute-bound RNAs-CrossLinking ImmunoPrecipitation (CLEAR-CLIP) method was established and used to identify the AGO2-miRNA-mRNA chimera regulating early trophoblast differentiation. MiRNA-specific PCR followed by next-generation sequencing identified FZD4 and SNN as the mRNA targets of TSC-specific miRNAs miR-516b-5p and 517b-3p, respectively. The regulations of the two miRNAs on the target gene expressions were further confirmed by the treatment of antagomirs. Higher levels of FZD4 and SNN were also found in AME lineage, suggesting the role of AGO2-miRNA in suppressing AME-related genes during trophoblast development. In conclusion, this study offered valuable insights into the functions of DNA methylation and AGO2-miRNA complex during trophoblast differentiation, which can enhance our comprehension of trophoblast development in early human embryos, and potentially facilitate the exploration of effective strategies for predicting and intervening placenta-related pregnancy complications.