The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, gamma-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and VEGF. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro . Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development as well as disease modeling and drug screening.

The TGFβ pathway plays an essential role in embryonic development, organ homeostasis, tissue repair, and disease1,2. This diversity of tasks is achieved through the intracellular effector SMAD2/3, whose canonical function is to control activity of target genes by interacting with transcriptional regulators3. Nevertheless, a complete description of the factors interacting with SMAD2/3 in any given cell type is still lacking. Here we address this limitation by describing the interactome of SMAD2/3 in human pluripotent stem cells (hPSCs). This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which deposits N6-methyladenosine (m6A)4. We uncover that SMAD2/3 promotes binding of the m6A methyltransferase complex onto a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor NANOG, thereby poising them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulations of the epitranscriptome. These novel aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer5.

SummaryDespite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR–Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR–Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.