Oral 1 - 1.3
1,2,3,4Emily McGaugh, 5Simon Kelley, 6April Craft, 1,2,3,4Maria Cristina Nostro
1 Toronto General Hospital Research Institute; 2 University Health Network; 3 McEwen Centre for Regenerative Medicine; 4 Physiology Department, University of Toronto; 5 SickKids, Toronto; 6 Boston Children’s Hospital
Introduction: Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of the insulin producing β cells located within the pancreas. The most common treatment for T1D patients is the administration of exogenous insulin, typically through the use of injections or an insulin pump. Despite improvements in T1D technology, patients continue to have a difficult time regulating their blood glucose and suffer from complications associated with the disease including, kidney disease, heart disease and even death. Human embryonic stem cells (HESC) offer a promising therapy for T1D patients, as they can be differentiated in vitro to pancreatic cells, offering an unlimited supply of cells for therapy. HESC can be differentiated to pancreatic progenitors (PPs) in vitro using a 4-stage differentiation protocol. Upon transplantation in mice, PPs can generate all cells of the pancreas including functional beta cells, and can restore normoglycemia in an STZ-induced diabetic mouse model. PPs can also be differentiated to insulin producing β-like cells in vitro using a 7-stage protocol. Generating mature and functional beta cells, however, has proven to be challenging as the final maturation of these cells only occurs in vivo. This suggests that the signaling pathways guiding pancreas development are not understood. Thus, the goal of my project is to identify signalling pathways critical for pancreas development in order to generate mature β-cells in vitro.
Hypothesis/Rationale: To identify signalling pathways that are important for pancreas development, we performed a mass spectrometry analysis to identify cell surface markers expressed by PPs compared to undifferentiated HESC and polyhormonal (PH) cells, a type of pancreatic cell that will not form a beta cell upon transplantation in mice. One marker identified as being exclusively expressed in PPs was FGFR3, a fibroblast growth factor (FGF) receptor. FGF ligands are expressed in most tissues and have key roles during early embryonic development and organogenesis, particularly during endoderm patterning and the development of organs such as the intestine, lung and pancreas. Remarkably, studies in the mouse embryo demonstrated that signaling through Fgfr3 inhibits expansion of the pancreatic epithelium. Although FGF signaling has been extensively studied in mice and other organisms, its role in human development is unknown. What has been shown, however, is that a hyperactive FGFR3 mutation in humans is known to lead to achondroplasia, a disease characterized by skeletal abnormalities and is the main form of dwarfism. Pancreatic function within this population has not been fully studied, however glucose tolerance tests performed on patients with achondroplasia show discordant results, indicating more investigation is needed. Based on this data, I hypothesize that FGFR3 inhibits human pancreatic development and by using FGFR3 inhibitors at specific developmental stages I can improve β-cell differentiation.
Methods/Results: Flow cytometry and qPCR analysis confirmed the mass spectrometry data showing higher expression of FGFR3 in PPs compared to hESCs and PH cells. Preliminary data showed that upon FGFR3 stimulation using two known FGFR3 ligands, FGF2 and FGF4, the percentage of PPs was decreased. The percentage of PPs could however be restored upon treatment with an FGFR3 inhibitor, suggesting that FGFR3 may inhibit human pancreas development. I will use CRISPR-Cas9 to knock out FGFR3 during differentiation, in order to identify the specific role of FGFR3 during pancreatic development. Additionally, I have already expanded and begun differentiating an induced pluripotent stem cell (iPSC) line derived from a patient with achondroplasia, having a hyperactive FGFR3 gene. By differentiating these iPSCs to β-like cells, I will be able to determine the effect FGFR3 hyper activation has on pancreas development. This will be very interesting, as activation studies done thus far have been limited by the ability of ligands to bind other FGF receptors. Through this I will not only enhance my understanding of how FGFR3 impacts key stages of β-cell development, but determine if patients with FGFR3 mutations are more likely to suffer from defects in carbohydrate metabolism, something not clearly understood. I will use this information to enhance our differentiation protocol and test the effect FGFR3 inhibition and stimulation has on beta cell maturation.
Discussion: In conclusion, FGFR3 is highly expressed in PPs and manipulation of FGF singling impacts human pancreatic induction. With the literature and preliminary data suggesting a role for FGF signaling in pancreas development, I believe that by further elucidating this signaling pathway I will be able to enhance our protocol ands generate a mature β cell in vitro.