Human NKX2.2 influences islet endocrine cell fate choices through regulation of WNT pathway genes

SUMMARY Transcriptional regulation is a key central mechanism of cell fate determination in developing tissues. The homeobox transcription factor NKX2.2 is an essential regulator of mouse and human pancreatic endocrine development, however its precise molecular role in a human system has not been previously investigated. In this study we generated NKX2.2 null (NKX2.2KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9 technologies and differentiated them towards a pancreatic β cell fate using a stem cell-derived β cell differentiation protocol. Functional and transcriptomic analyses of the hESC-derived pancreatic endocrine cells lacking NKX2.2 revealed similarities and differences compared to the molecular functions of NKX2.2 in mice. In the absence of NKX2.2, the β cell differentiations result in reduced numbers of insulin-producing cells, and the differentiations become skewed towards polyhormonal fates, including cells co-expressing insulin, ghrelin and somatostatin. Deletion of NKX2.2 also eliminates the off-target formation of enterochromaffin cells. Single cell transcriptome analysis of the early endocrine cell population revealed a marked disruption of metabolic pathways that was confirmed by comparative metabolite tracing, providing novel insights into the regulation of early endocrine lineage decisions. Furthermore, NKX2.2 directly regulates several genes in the WNT signaling pathway, suggesting this is a key molecular mechanism through which NKX2.2 regulates these islet cell fate decisions in the human system.