AbstractThe generation of definitive endoderm (DE) cells in sufficient numbers is a prerequisite for cell-replacement therapy for liver and pancreatic diseases. Previously, we reported that human adipose-derived mesenchymal stem cells (hAMSCs) can be induced to DE lineages and subsequent functional cells. Clarifying the regulatory mechanisms underlying the fate conversion from hAMSCs to DE is helpful for developing new strategies to improve the differentiation efficiency from hAMSCs to DE organs. Long noncoding RNAs (lncRNAs) have been shown to play pivotal roles in developmental processes, including cell fate determination and differentiation. In this study, we profiled the expression changes of lncRNAs and found that antidifferentiation noncoding RNA (ANCR) was downregulated during the differentiation of both hAMSCs and embryonic stem cells (ESCs) to DE cells. ANCR knockdown resulted in the elevated expression of DE markers in hAMSCs, but not in ESCs. ANCR overexpression reduced the efficiency of hAMSCs to differentiate into DE cells. Inhibitor of DNA binding 2 (ID2) was notably downregulated after ANCR knockdown. ID2 knockdown enhanced DE differentiation, whereas overexpression of ID2 impaired this process in hAMSCs. ANCR interacts with RNA-binding polypyrimidine tract-binding protein 1 (PTBP1) to facilitate its association with ID2 mRNA, leading to increased ID2 mRNA stability. Thus, the ANCR/PTBP1/ID2 network restricts the differentiation of hAMSCs toward DE. Our work highlights the inherent discrepancies between hAMSCs and ESCs. Defining hAMSC-specific signaling pathways might be important for designing optimal differentiation protocols for directing hAMSCs toward DE.