Developing a human-iPSC skeletal muscle model of glucose metabolism on responsive elastomer nanofibers

Diabetes affects close to 5 million people in the UK, a number that is likely to increase in the next decade due to an increasing rate of obesity. People with diabetes are unable to regulate their blood sugar levels, which are normally controlled by the hormone insulin. High blood glucose as a result of decreased uptake into cells can result in serious medical complication, including blindness, strokes, and necrosis of limbs. Insulin is produced in the pancreas, and induces cells of the body, primarily muscle cells, to take up glucose and re-balance sugar levels following meals. However, in people developing type-2 diabetes, skeletal muscle no longer responds properly to insulin - a condition known as insulin resistance. While diabetes can be treated with insulin injections, in type-2 diabetes, the most common form of diabetes, insulin resistance renders this therapy ineffective over time. Therefore, a more thorough understanding of the molecular pathways involved in insulin resistance would greatly facilitate the development of new treatments of diabetes.

Culturing human cells in a dish provides an ideal platform for finding new drugs, as this avoids issues with species differences, ethical considerations and high costs associated with experimental animals. Cultured skeletal myofibers would be an ideal for studying glucose metabolism, because this tissue is responsible for ca. 80-90% of glucose uptake following meals. However, to date, their use in diabetes modelling has been hampered by the fact that they are contractile, and detach quickly from rigid plastic surfaces in conventional cell culture. In addition, myofibers require innervation by nerve cells called motor neurons to mature into the equivalent of adult human muscle. Researchers have used fat cells to model diabetes instead, but these cells differ in how they control glucose uptake, in particular with regards to cellular trafficking of the glucose transporter GLUT4, a key factor in the regulation of blood sugar by insulin.

Here, we propose to fill this technological gap in diabetes research by adapting a 3D-coculture system developed by members of our team for neuromuscular disease studies to investigate insulin responses in muscle. The culture system combines motor neurons and myofibers derived from human induced pluripotent stem cell (hiPSCs) into a multi-well device suitable for high content imaging, a technology commonly used for drug screens. In the devices, muscle fibres are stabilized by a scaffold of aligned elastic nanofibers, which guide uniform growth of myofibers and prevent collapse. We will equip culture wells with nanofiber-based glucose biosensors to directly measure changes in glucose levels. We will incorporate genetic probes to aid the analysis: Motor neurons will be genetically engineered such that their activity can be controlled by light. Myofibers will carry a fusion gene of GLUT4 and red fluorescent protein, which will allow us to track the movement of GLUT4 in live cells in response to insulin and/or exercise.

Our proposal combines microdevice manufacturing, hiPSC-derivation of tissue, and analysis of metabolic pathways into a new neuromuscular culture model of diabetes. By the end of the project, we will have established the neuron/myofiber co-culture system, shown that innervated skeletal muscle myofibers take up glucose and mimic insulin responses, and recapitulate the failure of these processes in diabetes. We will have carried out a proof-of-principle screen with potential pharmacological treatments to show the suitability of the system for future drug discovery.