Abstract The greatest challenge to improving human stem cell derived β (SC-β) cell technologies and in turn impeding advancements in diabetes cell therapeutics and disease modeling is our lack of knowledge on human β cell functional maturation. In my recently published first author publication I develop a protocol for the generation of SC-β cells with insulin secretion dynamics approaching that of primary human islets and reveal that permitting TGFβ signaling is important for SC-β cell functional maturation. In additional preliminary data, I have identified that SIX2 is upregulated in TGFβ permitted cells and that SIX2 is necessary for SC-β cell function. In this project I will investigate TGFβ signaling and SIX2 expression in the context of SC-β differentiation and functional maturation. I hypothesize that TGFβ signaling via SIX2 transcriptional activity is critical for SC-β cell development and functional maturation. An understanding of TGFβ signaling in β cell functional maturation is not clear, with limited literature on the topic and the data being confounding and often contradictory with some reports claiming TGFβ signaling benefits and other harm β cell function. This proposal will help clarify the ambiguity associated with TGFβ and functional maturation. SIX2 is a recently identified β cell specific transcription factor within the islet of Langerhans and is linked to diabetes by GWAS studies. The regulatory role of SIX2 in pancreatic and β cell development and functional maturation has not been characterized. SIX2 is not expressed in mouse β cells making our SC-β cell differentiation model critical for investigating its role in pancreatic and β cell differentiation and functional maturation. To investigate the role of TGFβ signaling in SC- β cell functional maturation I will determine which SMADS and TGFβ ligands drive the functional maturation observed as well as the downstream mechanism driving the functional maturation using a combination of knockdown, overexpression, and RNA sequencing techniques. To investigate SIX2 action in SC-β cell differentiation and functional maturation I will determine SIX2 expressing populations through the differentiation using immunostaining and a SIX2 knockout human pluripotent stem cell line. I will investigate SIX2 role on SC- β cell functional maturation using RNA sequencing techniques and robust functional characterization employing calcium imaging, transmission electron microscopy, and insulin/proinsulin protein content assays. This work will elucidate the role of TGFβ and SIX2 on SC-β cell functional maturation. Aim 1. Investigate TGFβ signaling in SC-β cell functional maturation. Aim 2. Characterize SIX2 in SC-β cell differentiation and functional maturation.