scRNA-seq and snATAC-seq reveal differentiation trajectories of several pancreatic islet induction protocols
With the help of scRNA-seq and snATAC-seq, we unraveled the differentiation trajectories and gene co-expression networks of current islet induction protocols. Inspired by our understanding to in vivo pancreatogenesis, we improved the current protocol by inducing human anterior lip endoderm at stage 2 and promoting ventral pancreas fate. This refined differentiation protocol simplifies the existing multistage processes and generates higher-quality sc-islets with larger beta cell proportion and enhanced function of glucose regulation, which are validated by scRNA-seq and snATAC-seq. Overall design: Single cells were collected from the culturing system of Protocol A, B and C in different stages and sequenced following the Smartseq3 protocol. Protocol A and B are current islet induction protocols, while Protocol C is the refined protocol rised from this article. To investigate the effects of detail conditions implemented in Protocol C, we also leveraged Smartseq3 to optimize the small molecules' concentration and acting duration. Furthermore, we performed snATAC-seq to study the epigenetic differences between Protocol A and C. We examined the robustness of Protocol C's improvement by applying our induction systerm to human IPS cell line (hIPSC). Smartseq3 scRNA-seq showed the Protocol C's superiority maintained in hIPSC-induced sc-islets. Finally, we transplanted Protocol A and C generated sc-islets into diabetic mice. The 30 days post-transplanted grafts were collected and sequenced following the Smartseq3 protocol, that showed the Protocol C's grafts contained larger proportion of sc-beta cells.