The greatest challenge to improving human stem cell derived ? (SC-?) cell technologies and in turn impeding advancements in diabetes cell therapeutics and disease modeling is our lack of knowledge on human ? cell functional maturation. In my recently published first author publication I develop a protocol for the generation of SC-? cells with insulin secretion dynamics approaching that of primary human islets and reveal that permitting TGF? signaling is important for SC-? cell functional maturation. In additional preliminary data, I have identified that SIX2 is upregulated in TGF? permitted cells and that SIX2 is necessary for SC-? cell function. In this project I will investigate TGF? signaling and SIX2 expression in the context of SC-? differentiation and functional maturation. I hypothesize that TGF? signaling via SIX2 transcriptional activity is critical for SC-? cell development and functional maturation. An understanding of TGF? signaling in ? cell functional maturation is not clear, with limited literature on the topic and the data being confounding and often contradictory with some reports claiming TGF? signaling benefits and other harm ? cell function. This proposal will help clarify the ambiguity associated with TGF? and functional maturation. SIX2 is a recently identified ? 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 ? cell development and functional maturation has not been characterized. SIX2 is not expressed in mouse ? cells making our SC-? cell differentiation model critical for investigating its role in pancreatic and ? cell differentiation and functional maturation. To investigate the role of TGF? signaling in SC- ? cell functional maturation I will determine which SMADS and TGF? 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-? 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- ? 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 TGF? and SIX2 on SC-? cell functional maturation.
Aim 1. Investigate TGF? signaling in SC-? cell functional maturation.
Aim 2. Characterize SIX2 in SC-? cell differentiation and functional maturation.
Human pluripotent stem cell derived ? (SC-?) cells have immense potential for diabetes cell replacement therapy. Current SC-? cells have lower function than primary ? cells. This project aims to understand how TGF? signaling and SIX2 expression regulate SC-? cell functional maturation with aims to improve SC-? cell function. Improved SC-? cells will facilitate diabetes cell therapy, disease modeling, and drug screening.
