Investigating genetic and transcriptional regulation of human islet cells

Glucose metabolism and homeostasis, maintained within a tightly controlled euglycemic range, are critical for the proper function of many organs and tissues within organisms throughout the animal kingdom. The islets of Langerhans, which are endocrine micro-organs dispersed throughout the pancreas, are crucial for these processes. Composed of heterogeneous cell types, islets sense glucose levels and release hormones that direct target tissues to use, produce, and store glucose as needed. Two of these islet cell types, namely beta and alpha cells, work in tandem to regulate the delicate balance of euglycemia through the production of counterregulatory hormones. Beta cells secrete insulin, which lowers circulating blood glucose levels during fed or high glucose states, while alpha cells secrete glucagon, which raises blood glucose levels during fasted or low glucose conditions. Diabetes, including type 1 (T1D), type 2 (T2D), and monogenic forms, are characterized in part by islet dysfunction and dysregulated insulin and glucagon secretion. While both alpha and beta cells are essential to the mechanisms underlying diabetes pathophysiology, comparatively little is known about alpha cells in contrast to the well-studied beta cells, particularly regarding the regulation of hormone secretion. The gap in knowledge between beta and alpha cells is partly due to technical limitations that hinder the study of genes affecting alpha cell function in the same comprehensive manner as beta cells. For example, regulatory X box-binding factor 6 (RFX6) is a transcription factor that is expressed in both alpha and beta cells, with higher expression in alpha cells. Mutations of the gene are associated with various forms of diabetes. While our understanding of RFX6 in beta cell identity and insulin secretion has been extensively characterized using mouse and cell line models, RFX6 has not been studied in mature alpha cells. However, challenges that preclude the study of RFX6 in mature alpha cells include postnatal lethality in homozygous mice, failure to recapitulate RFX6 haploinsufficiency phenotypes, and lack of a human alpha cell line. Additionally, there is currently no efficient method to target specific cell types, such as alpha cells, for genetic manipulation in primary human islet models, and indistinguishable human and mouse glucagon obstruct alpha cell transplantation studies. Given these obstacles, there is a pressing need to develop models that allow for the study of alpha cell mechanisms underlying diabetes, not only focusing on RFX6, but also to gain a better understanding of alpha cell function in general to advance our knowledge of diabetes pathophysiology and pave the way for more effective treatments and interventions. In this thesis, we present primary human and mouse models to investigate the genetic and transcriptional regulation of human islet cells, overcoming some of the limitations outlined above. In Chapter 2, we describe the state of the field in which reaggregated primary human islets, or "pseudoislets", can be made by dispersing native islets and possess the unique ability to undergo efficient genetic manipulation while maintaining their hallmark features. In Chapter 3, we build upon the pseudoislet platform to be able to enrich for and genetically manipulate alpha cells to interrogate and identify novel functions of the transcription factor RFX6 in adult human alpha cells. Finally, in Chapter 4, we present the preliminary development of tools for use with the pseudoislet model, including enrichment for primary human alpha cells, genetic manipulation of more than one target with lentiviral transduction, and assessment of endocrine/enteroendocrine genes in an intestinal organoid model. Collectively, these studies demonstrate the value of human islet-based models in investigating human islet cell function and revealing insights that would not otherwise be uncovered in other model systems, particularly in lesser studied cell types like alpha cells.

Keywords: Medicine.
Developmental Biology.
Advisor: Kim Seung K.
Gloyn Anna L.
Nusse Roel
Svensson Katrin J.
Talbot William