2026-LB: Investigating the Role of NKX6.1 in Pancreatic Endocrine Cell Differentiation

Protocols to generate stem cell-derived beta cells have allowed novel investigations into the differentiation, specification and function of human beta cells. The transcription factor Nkx6.1 is critical to the specification of insulin-producing pancreatic β cells in mice. Knockout of Nkx6.1 in an inducible mouse model has been shown to demonstrated lack of β cell specification when Nkx6.1 is absent and instead a preferred differentiation to α cells. While Nkx6.1 is a highly conserved transcription factor between mice and humans, the role of NKX6.1 in human endocrine specification has not been studied in detail. Using CRISPR/CAS9 genome engineering, we generated an NKX6.1 knockout (KO) embryonic stem cell line to allow us to investigate NKX6.1 in the human context. Utilizing a directed differentiation protocol to produce stem-cell derived pancreatic β cells (sBCs), we hypothesize that the lack of NKX6.1 will induce a preference for α cell specification and eliminate β cell fate in NKX6.1 KO cells but not control wild-type (WT) cells, similar to what is observed in rodents. In addition, we will use an α-like cell differentiation protocol to test if α cell differentiation is altered between NKX6.1 KO and WT cells. Our experiments will provide genetic evidence that NKX6.1 is necessary for the specification of pancreatic β cells in humans and more fully elucidate the role of NKX6.1 in pancreatic endocrine cell fate.