Objective
The primary objective of this research is to advance our understanding of the mechanisms that underlie enhanced beta cell function and how deregulation of these mechanisms may lead to a loss of function. To do so I will use human stem cell-derived beta (sc-beta) cells to model human beta cell functional maturation and dysfunction via the expression of the MAFA transcription factor. In addition, I will analyze how a more stable mutant version of MAFA that compromises beta cell function and identity. I anticipate that these efforts will reveal novel insights into how beta cell activity can be restored in patients with T1D and enhanced upon transplantation of sc-beta cells.
Background Rationale
Improving upon Type 1 Diabetes therapies requires a better understanding of the cellular processes that regulate beta cell function and how their perturbation contributes to a loss of function. Sc-beta cells afford us the opportunity to model these processes which would otherwise not be possible.
Current state-of-the-art sc-beta cell differentiation protocols are generating sc-beta cells that more closely resemble their primary human beta cell counterparts than ever before. However, a key beta cell gene that continues to be expressed at low levels in sc-beta cells is MAFA. In humans, MAFA expression is increased around 9 years of age and it is believed to be integral for enhancing beta cell metabolic activity and insulin secretion that continues into adulthood. Increasing MAFA expression is predicted to improve the quality of sc-beta cells produced in the lab. In stark contrast, a stable MAFA S64F pathological variant found in human populations has been shown to cause beta cell dysfunction and predispose individuals to diabetes or insulinomatosis. These findings suggest that MAFA activity must be finely tuned to elicit its functional benefits. In my initial studies I have generated sc-beta cells capable of overexpressing MAFA WT or MAFA S64F. Consistent with MAFA being a critical beta cell maturation factor, I find that MAFA WT sc-beta cells have increased metabolic activity and glucose-stimulated insulin secretion. Interestingly, the MAFA S64F sc-beta cells show a transient increase in insulin secretion functionality before becoming non-functional. I pose that using MAFA WT and MAFA S64F scbeta cells to define the positive vs destructive activities, respectively, will allow the generation of sc-derived cells that display enhanced beta cell functional properties.
Description of Project
During the pathology of Type 1 Diabetes beta cell destruction results in a lack of insulin and subsequently high blood glucose levels. To improve Type 1 Diabetes patients’ quality of life, it is pivotal that new therapies be developed to prevent a loss of functional beta cells or to replace them. Human stem cell-derived beta (sc-beta) cells are invaluable tools for studying beta cell biology as well as a promising limitless source for cell replacement therapies. Extraordinary progress has been made in generating lab-made sc-beta cells that secrete insulin in response to glucose. However, discrepancies in gene expression and glucose metabolism persist between functionally mature human beta cells and sc-beta cells. An important beta cell gene that is expressed at low levels in sc-beta cells is MAFA. MAFA is a transcription factor that is involved in enhancing human beta cell glucose metabolism and acquisition of full functional properties. I have generated preliminary data indicating that the level of MAFA activity has to be tightly regulated to permit beta cell function and stability. In this proposal I will follow up on these results and induce expression of MAFA in sc-beta cells to mechanistically determine how MAFA drives beta cell maturation. Moreover, alongside wildtype MAFA, I will also overexpress a naturally occurring human mutant variant of MAFA with increased activity that is known to cause beta cell dysfunction and lead to the development of diabetes and insulinomatosis in human patients. The findings from these studies will advance our knowledge of the mechanisms that govern beta cell function and how deregulation of these mechanisms leads to beta cell dysfunction. Not only will this work have significant implications for therapies that aim to prevent beta cell dysfunction, but also for improving sc-beta cells as a cell replacement therapy.
Anticipated Outcome
I anticipate that these studies will reveal critical insight into the long sought after MAFA mechanisms that promote beta cell functional maturation. While MAFA activity in beta cells is generally held to be beneficial, the discovery of the overactive mutant version of MAFA S64F, that is detrimental to beta cell function, suggests that the consequences of MAFA activity are more nuanced. Therefore, including the MAFA S64F mutant in these studies will have the added benefit of uncovering how different levels of MAFA activity impacts beta cell processes and how this translates into beta cell functionality.
Relevance to T1D
This research is highly relevant to Type 1 Diabetes by increasing our understanding of human beta cell functional maturation and dysfunction. These studies will optimize functional properties of sc-beta cells and further establish them as a valuable resource for modeling human beta cell development and disease. The findings from this research will have significant implications for the development of therapies that target the prevention of beta cell dysfunction as well as improving sc-beta cells for cell replacement therapies.