Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naı ¨ve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placentaderived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Trophoblast are extra-embryonic cells that are essential to maintain pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT) composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naïve PSC-derived TE and CT (nCT) recreated the human and monkey TE-to-CT transition. nCT self-renewed as CT stem cells and had the characteristics of proliferating villous CT and CT in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (pBAP), pBAP were distinct from nCT and human placenta-derived CT stem cells, exhibiting properties consistent of the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naïve human PSCs. Our system will provide a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.

The faithful shutdown of the somatic program occurs in the early stage of reprogramming. Here, we examined the effect of in vivo reprogramming on Kras-induced cancer development. We show that the transient expression of reprogramming factors (1–3 days) in pancreatic acinar cells results in the transient repression of acinar cell enhancers, which are similarly observed in pancreatitis. We next demonstrate that Kras and p53 mutations are insufficient to induce ERK signaling in the pancreas. Notably, the transient expression of reprogramming factors in Kras mutant mice is sufficient to induce the robust and persistent activation of ERK signaling in acinar cells and rapid formation of pancreatic ductal adenocarcinoma. In contrast, the forced expression of acinar cell-related transcription factors inhibits the pancreatitis-induced activation of ERK signaling and development of precancerous lesions in Kras-mutated acinar cells. These results underscore a crucial role of dedifferentiation-associated epigenetic regulations in the initiation of pancreatic cancers.