Real-time intracellular monitoring of microRNAs in human stem cell-derived insulin producing organoids

Project Summary/Abstract Aside from daily Insulin therapy, intra-portal hepatic transplantation of cadaveric donor islets is the only other recourse for patients with type 1 diabetes (T1D) for long-term management of this disease. Exogenous administration of Insulin does not replicate what the endogenous beta cell, an exquisite sensor and regulator of circulating blood glucose, accomplishes so elegantly. Replacement therapy currently suffers from limited access to primary islets for grafting, and the immediate damage to the graft post-transplantation due to insufficient vascularization and hypoxia. Furthermore, current transplantation success is variable, with C- peptide levels and glucose measurements used as readouts of cell function, parameters that have a delayed onset. Real-time monitoring of cell health and function in recipients has remained elusive. Here, we describe novel sensors with the capability to detect intracellularly changes for e.g. responding to a stressful stimulus, and transmit a measurable signal in real-time to communicate such specific molecular changes with high sensitivity. Human stem cells are described as a limitless source of cells for replacement therapy, and have been under intense scrutiny and investigation over the last several decades. Recent success in generating pancreatic cells including the Insulin-producing beta cell from human stem cells heralds a new era in regenerative medicine, simultaneously representing material that serves as a surrogate for cadaveric islets, and a platform to develop tools that can report the health of cell grafts post-transplantation, a technology that is missing from current methods to evaluate cell health. To develop such a detection system to assess viability and health of beta cell organoids, we have engineered nanoprobes that bind microRNAs (miRNAs) in cell extracts and intracellularly, and serve as a homing beacon to identify endogenous RNA species without pre-amplification. MicroRNAs are important regulators of gene expression and are known to be dysregulated in disease, serving as unique diagnostic markers for sensing cell health. Our nanoprobes are specific, sensitive, and can be detected in vivo in animal models. Here, we merge these two technologies to tackle the challenge of monitoring tissue health in graft recipients suffering from T1D. Beta cell clusters/organoids that we generate in a dish will incorporate specific sensors that detect microRNA changes triggered by the onset of hypoxia. Hypoxia is a major roadblock facing islet transplantation approaches today. Our long-term goal is to develop tools that can sense, in vivo, compromised graft health long before secondary readouts such as reduced C-peptide levels and dysglycemia, and improve transplantation success for patients with T1D.Project Narrative The success of beta cell replacement therapy for patients with type 1 diabetes (T1D) suffers from a paucity of donor islets, an inhospitable transplant environment that causes graft failure, and the absence of sensing technology capable of monitoring cell health, function, and duress. We address these challenges by generating surrogate islet organoids from stem cells and engineering them to sense small RNA species that are molecular biomarkers for cellular dysfunction. Our proposal describes novel strategies for non-invasive assessment of cellular health and viability within beta cell organoids as a response to environmental stress that have direct implications for long-term survival and proper function of islet grafts, leading to insulin independence for patients with T1D.