N 6 -methyladenosine modification-mediated mRNA metabolism is essential for human pancreatic lineage specification and islet organogenesis.

Pancreatic differentiation from human pluripotent stem cells (hPSCs) provides promising avenues for investigating development and treating diseases. N 6 -methyladenosine (m 6 A) is the most prevalent internal messenger RNA (mRNA) modification and plays pivotal roles in regulation of mRNA metabolism, while its functions remain elusive. Here, we profile the dynamic landscapes of m 6 A transcriptome-wide during pancreatic differentiation. Next, we generate knockout hPSC lines of the major m 6 A demethylase ALKBH5, and find that ALKBH5 plays significant regulatory roles in pancreatic organogenesis. Mechanistic studies reveal that ALKBH5 deficiency reduces the mRNA stability of key pancreatic transcription factors in an m 6 A and YTHDF2-dependent manner. We further identify that ALKBH5 cofactor α-ketoglutarate can be applied to enhance differentiation. Collectively, our findings identify ALKBH5 as an essential regulator of pancreatic differentiation and highlight that m 6 A modification-mediated mRNA metabolism presents an important layer of regulation during cell-fate specification and holds great potentials for translational applications.